Novel coelenterazine substrates and methods of use

ABSTRACT

An isolated polynucleotide encoding a modified luciferase polypeptide and novel coelenterazine-based substrates. The OgLuc variant polypeptide has at least 60% amino acid sequence identity to SEQ ID NO: 1 and at least one amino acid substitution at a position corresponding to an amino acid in SEQ ID NO: 1. The OgLuc variant polypeptide has at least one of enhanced luminescence, enhanced signal stability, and enhanced protein stability relative to the corresponding polypeptide of the wild-type  Oplophorus  luciferase.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application No.61/409,422, filed Nov. 2, 2010, which is incorporated herein byreference in its entirety.

BACKGROUND

The luciferase secreted from the deep-sea shrimp Oplophorusgracilirostris has been shown to possess many interestingcharacteristics, such as high activity, high quantum yield, and broadsubstrate specificity (including, e.g., coelenterazine as well asvarious coelenterazine analogs). The bioluminescent reaction ofOplophorus takes place when the oxidation of coelenterazine (substrate)with molecular oxygen is catalyzed by Oplophorus luciferase, resultingin light of maximum intensity at 462 nm and the products CO₂ andcoelenteramide (Shimomura et al., Biochemistry, 17:994 (1978)). Optimumluminescence occurs at pH 9 in the presence of 0.05-0.1 M NaCl at 40°C., and, due to the unusual resistance of this enzyme to heat, visibleluminescence occurs at temperatures above 50° C. when the highlypurified enzyme is used or at over 70° C. when partially purified enzymeis used. At pH 8.7, the native luciferase was reported by Shimomura etal. (1978) to have a molecular weight of approximately 130 kDa,apparently comprising four monomers of 31 kDa each; at lower pH, thenative luciferase tends to polymerize.

Later work has shown that the Oplophorus gracilirostris luciferase is acomplex of native 35 kDa and 19 kDa proteins, i.e., a heterotetramerconsisting of two 19 kDa components and two 35 kDa components. Inouye etal. (2000) reported the molecular cloning of the cDNAs encoding the 35kDa and 19 kDa proteins of Oplophorus luciferase, and the identificationof the protein component that catalyzes the luminescence reaction. ThecDNAs encoding the proteins were expressed in bacterial and mammaliancells, and the 19 kDa protein was identified as the component capable ofcatalyzing the luminescent oxidation of coelenterazine (Inouye et al.,2000).

The 19 kDa protein of Oplophorus luciferase (GenBank accession BAB13776,196 amino acids) appears to be the smallest catalytic component havingluciferase function, and its primary structure has no significantsimilarity with any reported luciferase including imidazopyrazinoneluciferases (Lorenz et al., PNAS USA, 88:4438 (1991); Thompson et al.,PNAS USA, 86:6567 (1989)). Expression of the 19 kDa protein in E. coliresulted in the formation of inclusion bodies (Inouye and Sasaki,Protein Expression and Purification, 56:261-268 (2007)). The formationof inclusion bodies is likely due to the instability of the protein.

The substrate specificity of Oplophorus luciferase is unexpectedly broad(Inouye and Shimomura. BBRC, 223:349 (1997)). For instance,bisdeoxycoelenterazine (i.e., coelenterazine-hh), an analog ofcoelenterazine, is an excellent substrate for Oplophorus luciferasecomparable to coelenterazine (Nakamura et al., Tetrahedron Lett.,38:6405 (1997)). Moreover, Oplophorus luciferase is a secreted enzyme,like the luciferase of the marine ostracod Cypridina (Vargula)hilgendorfii (Johnson and Shimomura, Meth. Enzyme, 57:331 (1978)), whichalso uses an imidazopyrazinone-type luciferin to emit light.

SUMMARY

In an aspect, the disclosure relates to a compound of formula (Ia) or(Ib):

-   -   wherein R² is selected from the group consisting of

or C₂₋₅ straight chain alkyl;

-   -   R⁶ is selected from the group consisting of —H, —OH, —NH₂,        —OC(O)R or —OCH₂OC(O)R;    -   R⁸ is selected from the group consisting of

H or lower cycloalkyl;

-   -   wherein R³ and R⁴ are both H or both C₁₋₂ alkyl;    -   W is —NH₂, halo, —OH, —NHC(O)R, —CO₂R;    -   X is —S—, —O— or —NR²²—;    -   Y is —H, —OH, or —OR¹¹;    -   Z is —CH— or —N—;    -   each R¹¹ is independently —C(O)R″ or —CH₂OC(O)R″;    -   R²² is H, CH₃ or CH₂CH₃;    -   each R is independently C₁₋₇ straight-chain alkyl or C₁₋₇        branched alkyl;    -   R″ is C₁₋₇ straight-chain alkyl or C₁₋₇ branched alkyl;    -   the dashed bonds indicate the presence of an optional ring,        which may be saturated or unsaturated;    -   with the proviso that when R² is

or

R⁸ is not

with the proviso that when R² is

R⁸ is

or lower cycloalkyl; andwith the proviso that when R⁶ is NH₂, R² is,

or C₂₋₅ alkyl;

or R⁸ is not

In another aspect, the disclosure relates to a compound selected from

In an aspect, the disclosure relates to a compound of formula

In an aspect, the disclosure relates to an isolated polynucleotideencoding an OgLuc variant polypeptide having at least 60% amino acidsequence identity to SEQ ID NO: 1 comprising at least one amino acidsubstitution at a position corresponding to an amino acid in SEQ ID NO:1 wherein the OgLuc variant polypeptide has enhanced luminescence.

In an aspect, the disclosure relates to an isolated polynucleotideencoding an OgLuc variant polypeptide having at least 60% amino acidsequence identity to SEQ ID NO: 1 comprising at least one amino acidsubstitution at a position corresponding to an amino acid in SEQ ID NO:1 wherein the OgLuc variant polypeptide has enhanced luminescencerelative to an OgLuc polypeptide of SEQ ID NO: 3 with the proviso thatthe polypeptide encoded by the polynucleotide is not one of those listedin Table 47.

In an aspect, the disclosure relates to an isolated polynucleotideencoding an OgLuc variant polypeptide having at least 60% amino acidsequence identity to SEQ ID NO: 1 comprising at least one amino acidsubstitution at a position corresponding to an amino acid in SEQ ID NO:1 wherein the OgLuc variant polypeptide has enhanced luminescencerelative to a polypeptide of SEQ ID NO: 31 with the proviso that thepolypeptide encoded by the polynucleotide is not SEQ ID NO: 3 or 15.

In an aspect, the disclosure relates to an isolated polynucleotideencoding an OgLuc variant polypeptide having at least 60% amino acidsequence identity to SEQ ID NO: 1 comprising at least one amino acidsubstitution at a position corresponding to an amino acid in SEQ ID NO:1 wherein the OgLuc variant polypeptide has enhanced luminescencerelative to a polypeptide of SEQ ID NO: 29 with the proviso that thepolypeptide encoded by the polynucleotide is not SEQ ID NO: 3 or 15.

In an aspect, the disclosure relates to an isolated polynucleotideencoding an OgLuc variant polypeptide having at least 80% amino acidsequence identity to an OgLuc polypeptide of SEQ ID NO: 1 comprisingamino acid substitutions A4E, Q11R, A33K, V44I, P115E, Q124K, Y138I,N166R, I90V, F54I, Q18L, F68Y, L72Q, and M75K corresponding to SEQ IDNO: 1 and the OgLuc variant polypeptide having luciferase activity.

In an aspect, the disclosure relates to an isolated polynucleotideencoding an OgLuc variant polypeptide having at least 80% amino acidsequence identity to an OgLuc polypeptide of SEQ ID NO: 1, wherein theamino acid at position 4 is glutamate, at position 11 is arginine, atposition 18 is leucine, at position 33 is lysine, at position 44 isisoleucine, at position 54 is isoleucine; at position 68 is tyrosine, atposition 72 is glutamine, at position 75 is lysine, at position 90 isvaline, at position 115 is glutamate, at position 124 is lysine, atposition 138 is isoleucine, and at position 166 is argininecorresponding to SEQ ID NO: 1 and the OgLuc variant polypeptide havingluciferase activity.

In an aspect, the disclosure relates to an isolated polynucleotideencoding an OgLuc variant polypeptide having at least 80% amino acidsequence identity to an OgLuc polypeptide of SEQ ID NO: 1 comprisingamino acid substitutions A4E, Q11R, A33K, V44I, P115E, Q124K, Y138I,N166R, Q18L, F54I, L92H, and Y109F corresponding to SEQ ID NO: 1 and theOgLuc variant polypeptide having luciferase activity.

In an aspect, the disclosure relates to an isolated polynucleotideencoding an OgLuc variant polypeptide having at least 80% amino acidsequence identity to an OgLuc polypeptide of SEQ ID NO: 1 comprisingamino acid substitutions A4E, Q11R, A33K, V44I, A54I, F77Y, I90V, P115E,Q124K, Y138I and N166R corresponding to SEQ ID NO: 1 and the OgLucvariant polypeptide having luciferase activity.

In an aspect, the disclosure relates to an isolated polynucleotideencoding an OgLuc variant polypeptide having at least 80% amino acidsequence identity to an OgLuc polypeptide of SEQ ID NO: 1, wherein theamino acid at position 4 is glutamate, at position 11 is arginine, atposition 18 is leucine, at position 33 is lysine, at position 44 isisoleucine, at position 54 is isoleucine, at position 92 is histidine,at position 109 is phenylalanine, at position 115 is glutamate, atposition 124 is lysine, at position 138 is isoleucine, and at position166 is arginine corresponding to SEQ ID NO: 1 and the OgLuc variantpolypeptide having luciferase activity.

In an aspect, the disclosure relates to an isolated polynucleotideencoding an OgLuc variant polypeptide having at least 80% amino acidsequence identity to an OgLuc polypeptide of SEQ ID NO: 1, wherein theamino acid at position 4 is glutamate, at position 11 is arginine, atposition 33 is lysine, at position 44 is isoleucine, at position 54 isisoleucine, at position 77 is tyrosine, at position 90 is valine, atposition 115 is glutamate, at position 124 is lysine, at position 138 isisoleucine, and at position 166 is arginine corresponding to SEQ ID NO:1 and the OgLuc variant polypeptide having luciferase activity.

In an aspect, the disclosure relates to an isolated polynucleotidecomprising the polynucleotide encoding the polypeptide of SEQ ID NO: 19.

In an aspect, the disclosure relates to an isolated polynucleotidecomprising the polynucleotide of SEQ ID NO: 18, SEQ ID NO: 24, SEQ IDNO: 25, SEQ ID NO: 42, SEQ ID NO: 88, or SEQ ID NO: 92.

In an aspect, the disclosure relates to an isolated polynucleotideencoding a decapod luciferase polypeptide having at least 30% amino acidsequence identity to SEQ ID NO: 1, the polypeptide comprising a sequencepattern corresponding to the sequence pattern of Formula (VII) andincluding no more than 5 differences, wherein differences includedifferences from pattern positions 1, 2, 3, 5, 8, 10, 12, 14, 15, 17, or18 relative to Formula (VII) according to the OgLuc pattern listed inTable 4 as well as gaps or insertions between any of the patternpositions of Formula (VII) according to the OgLuc pattern listed inTable 4, wherein the decapod luciferase produces luminescence in thepresence of a coelenterazine.

In an aspect, the disclosure relates to a synthetic nucleotide sequenceencoding an OgLuc variant polypeptide comprising a fragment of at least100 nucleotides having 80% or less nucleic acid sequence identity to aparent nucleic acid sequence having SEQ ID NO: 2 and having 90% or morenucleic acid sequence identity to SEQ ID NO: 22, SEQ ID NO: 23, SEQ IDNO: 24, or SEQ ID NO: 25 or the complement thereof, wherein thedecreased sequence identity is a result of different codons in thesynthetic nucleotide sequence relative to the codons in the parentnucleic acid sequence, wherein the synthetic nucleotide sequence encodesa OgLuc variant which has at least 85% amino acid sequence identity tothe corresponding luciferase encoded by the parent nucleic acidsequence, and wherein the synthetic nucleotide sequence has a reducednumber of regulatory sequences relative to the parent nucleic acidsequence.

In an aspect, the disclosure relates to a synthetic nucleotide sequenceencoding an OgLuc variant polypeptide comprising a fragment of at least300 nucleotides having 80% or less nucleic acid sequence identity to aparent nucleic acid sequence having SEQ ID NO: 14 and having 90% or morenucleic acid sequence identity to SEQ ID NO: 22 or SEQ ID NO: 23 or thecomplement thereof, wherein the decreased sequence identity is a resultof different codons in the synthetic nucleotide sequence relative to thecodons in the parent nucleic acid sequence, wherein the syntheticnucleotide sequence encodes a firefly luciferase which has at least 85%amino acid sequence identity to the corresponding luciferase encoded bythe parent nucleic acid sequence, and wherein the synthetic nucleotidesequence has a reduced number of regulatory sequences relative to theparent nucleic acid sequence.

In an aspect, the disclosure relates to a synthetic nucleotide sequenceencoding an OgLuc variant polypeptide comprising a fragment of at least100 nucleotides having 80% or less nucleic acid sequence identity to aparent nucleic acid sequence having SEQ ID NO: 18 and having 90% or morenucleic acid sequence identity to SEQ ID NO: 24 or SEQ ID NO: 25 or thecomplement thereof, wherein the decreased sequence identity is a resultof different codons in the synthetic nucleotide sequence relative to thecodons in the parent nucleic acid sequence, wherein the syntheticnucleotide sequence encodes a OgLuc variant which has at least 85% aminoacid sequence identity to the corresponding luciferase encoded by theparent nucleic acid sequence, and wherein the synthetic nucleotidesequence has a reduced number of regulatory sequences relative to theparent nucleic acid sequence.

In an aspect, the disclosure relates to a fusion peptide comprising asignal peptide from Oplophorus gracilirostris fused to a heterologousprotein, wherein said signal peptide is SEQ ID NO: 54, wherein thefusion peptide is expressed in a cell and secreted from the cell.

In an aspect, the disclosure relates to a method of generating apolynucleotide encoding a OgLuc variant polypeptide comprising: (a)using a parental fusion protein construct comprising a parental OgLucpolypeptide and at least one heterologous polypeptide to generate alibrary of variant fusion proteins; and (b) screening the library for atleast one of enhanced luminescence, enhanced enzyme stability orenhanced biocompatibility relative to the parental fusion proteinconstruct.

In an aspect, the disclosure relates to a method of generatingcodon-optimized polynucleotides encoding a luciferase for use in anorganism, comprising: for each amino acid in the luciferase, randomlyselecting a codon from the two most commonly used codons used in theorganism to encode for the amino acid to produce a first codon-optimizedpolynucleotide.

Other aspects of the invention will become apparent by consideration ofthe detailed description and accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the chemical structure of native coelenterazine, knownbis-coelenterazine (coelenterazine-hh), and known coelenterazine-h,where R2, R6 and R8 represent the regions of the molecule wheremodifications were made.

FIG. 2 shows the chemical structure of novel coelenterazines PBI-3939,PBI-3889, PBI-3945, PBI-4002, PBI-3841, PBI-3897, PBI-3896, PBI-3925,PBI-3894, PBI-3932, and PBI-3840.

FIG. 3 shows the Km determination of PBI-3939.

FIG. 4 shows the chemical structure of various novel coelenterazines ofthe present invention.

FIGS. 5A-G show the luminescence (RLUs) generated from lysed bacterialcells expressing C1+A4E using native, known, and novel coelenterazine assubstrates. FIGS. 5A, 5C-5G show independent experiments measuring theluminescence in RLUs generated by C1+A4E with known and novelcoelenterazines using native coelenterazine as a comparison. FIG. 5Bshows the fold-decrease in luminescence generated by C1+4AE using thesubstrates shown in FIG. 5A compared to native coelenterazine.

FIGS. 6A-D show the luminescence generated from lysed bacterial cellsexpressing various OgLuc variants using native coelenterazine(“Coelente”), known coelenterazine-h (“h”), known coelenterazine-hh(“h,h”), known 2-methyl coelenterazine (“2-me”), known coelenterazine-v(“v”), and novel coelenterazines PBI-3840, PBI-3897, PBI-3889, PBI-3899,PBI-3900, PBI-3912, PBI-3913, PBI-3925, PBI-3897, PBI-3899, PBI-3889,PBI-3939, PBI-3933, PBI-3932, PBI-3946, PBI-3897, PBI-3841, PBI-3896,PBI-3925, and PBI-3945 as substrates.

FIG. 7 shows the amino acid substitutions in various OgLuc variants.

FIGS. 8A-B show the luminescence generated from lysed bacterial cellsexpressing OgLuc variants listed in FIG. 7 using native coelenterazine(“Coelenterazine”), known coelenterazine-h (“H”), knowncoelenterazine-hh (“h,h”), and novel coelenterazines PBI-3840, PBI-3925,PBI-3912, PBI-3889, PBI-3939, PBI-3933, PBI-3932, PBI-3946, PBI-3941,and PBI-3896 as substrates.

FIG. 9 shows the luminescence generated from lysed bacterial cellsexpressing various OgLuc variants using native coelenterazine(“Coelenterazine”), known coelenterazine-hh (“h,h”), and novelcoelenterazines PBI-3939, PBI-3945, PBI-3840, PBI-3932, PBI-3925,PBI-9894, and PBI-3896 as substrates.

FIG. 10 shows the amino acid substitutions in various OgLuc variants.

FIG. 11 shows the luminescence generated from lysed bacterial cellsexpressing OgLuc variants listed in FIG. 10 using native coelenterazine(“Coelenterazine”), known coelenterazine-hh (“h,h”), and novelcoelenterazines PBI-3939, PBI-3945, PBI-3840, PBI-3932, PBI-3925,PBI-3894, and PBI-3896, as substrates.

FIG. 12 shows the luminescence generated from lysed bacterial cellsexpressing OgLuc variants using native coelenterazine(“Coelenterazine”), known coelenterazine-hh (“h,h”), and novelcoelenterazines PBI-3939, PBI-3945, PBI-3889, PBI-3840, PBI-3932,PBI-3925, PBI-3894, PBI-3896, and PBI-3897 as substrates.

FIG. 13 shows the luminescence generated from lysed bacterial cellsexpressing OgLuc variants using native coelenterazine(“Coelenterazine”), known coelenterazine-hh (“H,H”), and novelcoelenterazines PBI-3897, PBI-3896, and PBI-3894 as substrates.

FIG. 14 shows the amino acid substitutions in various OgLuc variants.

FIG. 15 shows the luminescence generated from lysed bacterial cellsexpressing OgLuc variants listed in FIG. 14 using native coelenterazine(“Coelenterazine”), known coelenterazine-hh (“h,h”), and novelcoelenterazines PBI-3897, PBI-3841, PBI-3896, and PBI-3894 assubstrates.

FIG. 16 shows the luminescence generated from lysed bacterial cellsexpressing OgLuc variants using native coelenterazine(“Coelenterazine”), known coelenterazine-h (“H”), knowncoelenterazine-hh (“HH”), and novel coelenterazines PBI-3841 andPBI-3897 as substrates.

FIG. 17 shows the luminescence generated from lysed bacterial cellsexpressing various OgLuc variants and humanized Renilla luciferase (hRL)using native coelenterazine (“Coel”), known coelenterazine-hh (“h,h”),and novel coelenterazines PBI-3897 and PBI-3841 as substrates.

FIG. 18 shows the luminescence generated from lysed bacterial cellsexpressing various OgLuc variants using native coelenterazine(“Coelenterazine”), known coelenterazine-hh (“h,h”), and novelcoelenterazines PBI-3939, PBI-3945, PBI-3889, and PBI-4002 assubstrates.

FIG. 19 shows the luminescence generated from lysed bacterial cellsexpressing various OgLuc variants using native coelenterazine(“Coelenterazine”), known coelenterazine-h (“H”), knowncoelenterazine-hh (“h,h”), and novel coelenterazines PBI-3939, PBI-3945,PBI-3889, and PBI-4002 as substrates.

FIG. 20 shows the amino acid substitutions in various OgLuc variants.

FIG. 21 shows the luminescence generated from lysed bacterial cellsexpressing OgLuc variants listed in FIG. 20 using native coelenterazine(“Coelenterazine”), known coelenterazine-h (“H”), knowncoelenterazine-hh (“h,h”), and novel coelenterazines PBI-3939, PBI-3945,PBI-4002, PBI-3932, and PBI-3840 as substrates.

FIG. 22 shows the amino acid substitutions in various OgLuc variants.

FIG. 23 shows the luminescence generated from lysed bacterial cellsexpressing OgLuc variants listed in FIG. 22 using native coelenterazine(“Coelenterazine”), known coelenterazine-h (“H”), knowncoelenterazine-hh (“h,h”), and novel coelenterazines PBI-3939, PBI-3945,PBI-3889, PBI-4002, PBI-3932, and PBI-3840 as substrates.

FIG. 24 shows the luminescence generated from lysed bacterial cellsexpressing various OgLuc variants and hRL (“Renilla”) using nativecoelenterazine (“Coelenterazine”), known coelenterazine-h (“H”), knowncoelenterazine-hh (“H,H”), and novel coelenterazines PBI-3939 andPBI-3945 as substrates.

FIG. 25 shows the luminescence generated from lysed bacterial cellsexpressing various OgLuc variants and hRL (“Renilla”) using nativecoelenterazine (“Coelenterazine”), known coelenterazine-hh (“h,h”), andnovel coelenterazines PBI-3939, PBI-3945, PBI-3889, and PBI-4002 assubstrates.

FIG. 26 shows the amino acid substitutions in various OgLuc variants.

FIG. 27 shows the luminescence generated from lysed bacterial cellsexpressing OgLuc variants listed in FIG. 26 using native coelenterazine(“Coelenterazine”), known coelenterazine-h (“H”), knowncoelenterazine-hh (“h,h”), and novel coelenterazines PBI-3939, PBI-3945,PBI-3889, and PBI-4002 as substrates.

FIG. 28 shows the luminescence generated from lysed bacterial cellsexpressing various OgLuc variants and hRL (“Renilla”) using nativecoelenterazine (“Coel.”), known coelenterazine-h (“H”), knowncoelenterazine-hh (“H,H”), and novel coelenterazines PBI-3939, PBI-3945,PBI-3889, and PBI-4002 as substrates.

FIG. 29 shows the luminescence of 9B8 opt and 9B8 opt+K33N in bacteriallysates using native coelenterazine and PBI-3939 as substrates and therelative specificity of these variants for PBI-3939 compared to nativecoelenterazine.

FIGS. 30A-D show mutational analysis at position 166 using nativecoelenterazine (FIG. 30A), coelenterazine-h (FIG. 30B), and PBI-399(FIG. 30C).

FIG. 31 shows the luminescence of various deletions in the OgLuc variantL27V where (−) is the machine background.

FIG. 32 shows the normalized luminescence generated from lysed HEK293cells expressing hRL (“Renilla”) using native coelenterazine as asubstrate, firefly luciferase (Luc2) using luciferin (BRIGHT-GLO™ AssayReagent) as a substrate, and various OgLuc variants using novel PBI-3939as a substrate.

FIG. 33 shows the signal stability of IV and 15C1 in bacterial lysatesusing the novel coelenterazine PBI-3945 as a substrate and N and 9B8 inbacterial lysates using the novel coelenterazine PBI-3889 as asubstrate.

FIGS. 34A-B show the higher activity (FIG. 34A) and signal stability(FIG. 34B) of the OgLuc variant L27V compared to Firefly (Flue) andRenilla (Rluc) luciferases.

FIG. 35 shows the Vmax (RLU/sec) and Km (μM) values for various OgLucvariants in bacterial lysates using the novel coelenterazine PBI-3939 asa substrate.

FIG. 36 shows the Vmax (RLU/sec) and Km (μM) values for various OgLucvariants in bacterial lysates using the novel coelenterazine PBI-3939 asa substrate.

FIG. 37 shows the Vmax (RLU/sec) and Km (μM) values for 9B8 opt and 9B8opt+K33N both in bacterial lysates using the novel coelenterazinePBI-3939 as a substrate.

FIG. 38 shows the protein stability at 50° C. of various OgLuc variantsin bacterial lysates using native coelenterazine as a substrate as theluminescence at t=0 and half-life in min

FIGS. 39A-B show the structural integrity (determined by expression,stability, and solubility as shown by SDS-PAGE analysis) in bacteriallysates of various OgLuc variants at 25° C. (FIG. 39A) and 37° C. (FIG.39B) compared to Renilla (hRL) and firefly luciferase (Luc2).

FIGS. 40A-B show the protein stability at 60° C. in bacterial lysates of9B8 opt and 9B8 opt+K33N using the novel coelenterazine PBI-3939 as asubstrate as the natural log (ln) of the luminescence (in RLU) over time(FIG. 40A) and as the half-life in hrs (FIG. 40B).

FIG. 41 shows the percent activity of the OgLuc variants 9B8 and L27V at60° C.

FIGS. 42A-B show the protein stability of the OgLuc variant L27V atvarious pH (FIG. 42A) and salt concentrations (FIG. 42B).

FIGS. 43A-B shows the gel filtration chromatographic analysis ofpurified C1+A4E (FIG. 43A) and 9B8 (FIG. 43B).

FIG. 44 shows the gel filtration chromatographic analysis demonstratingthat the OgLuc variant L27V exists in a monomeric form.

FIGS. 45A-B show the protein expression levels of various OgLucvariant-HALOTAG® (HT7) fusion proteins in undiluted and 1:1 dilutedbacterial lysate samples analyzed by SDS-PAGE (FIG. 45A) and thenormalized protein expression levels (FIG. 45B).

FIGS. 46A-B show the protein expression (FIG. 46A) and solubility of theOgLuc variants 9B8 opt, V2 and L27V (FIG. 46B).

FIG. 47 shows the normalized luminescence in RLUs generated from lysedHEK293 cells expressing IV, 9B8, and hRL (“Renilla”) using nativecoelenterazine and the novel coelenterazine PBI-3939 as substrates.

FIG. 48 shows the normalized luminescence in RLUs generated from lysedHEK293 cells expressing pF4Ag-Ogluc-9B8-HT7, pF4Ag-Luc2-HT7 andpF4Ag-Renilla-HT7 using PBI-3939, Luciferin (BRIGHT-GLO™ Assay Reagent),and native coelenterazine, respectively, as a substrate.

FIG. 49 shows the luminescence generated from lysed HEK293 cellsexpressing 30 or 100 ng of plasmid DNA encoding either 9B8 opt or 9B8opt+K33N (“K33N”) using the novel coelenterazine PBI-3939 as asubstrate.

FIGS. 50A-E show the luminescence of the OgLuc variant L27V compared tofirefly luciferase (Luc2) in HEK 293 (FIG. 50A) and HeLa cells(non-fusion) (FIG. 50B), the luminescence of HaloTag® fusion compared tothe OgLuc variant L27V (FIG. 50C) and firefly luciferase (Luc2) (FIG.50D), and the protein expression of HaloTag®-OgLuc L27V compared toHaloTag®-Firefly luciferase (Luc2) in HEK 293 (“HEK”) and HeLa cells(“HeLa”).

FIG. 51 shows inhibition analysis of the OgLuc variants 9B8 and L27Vagainst a LOPAC library to determine their susceptibility to off-targetinteractions.

FIGS. 52A-E show the inhibition analysis of the OgLuc variants 9B8 andL27V by Suramin and Tyr ag 835 (FIGS. 52A-C) and the chemical structuresof Suramin (FIG. 52D) and Tyr ag 835 (FIG. 52E).

FIG. 53 shows the activity of the OgLuc variants 9B8 and L27V wasanalyzed in the presence of BSA to determine resistance to non-specificprotein interactions.

FIG. 54 shows the percent activity of the OgLuc variants 9B8 and L27V todetermine reactivity to plastic.

FIG. 55 shows the luminescence generated from lysed HEK293 cellsexpressing the IV cAMP transcriptional reporter compared to hRL(“Renilla”) using known coelenterazine-h as a substrate with (“induced”)or without (“basal”) forskolin treatment and the fold induction(response) due to forskolin treatment (“fold”).

FIG. 56 shows the normalized luminescence generated from lysed HEK293cells expressing the 9B8, 9B8 opt, hRL (“Renilla”) or firefly luciferase(“Luc2”) cAMP transcriptional reporter using PBI-3939 (for 9B8 and 9B8opt), native coelenterazine (for hRL) or luciferin (BRIGHT-GLO™ AssayReagent; for Luc2) as a substrate with (“+FSK”) or without (“−FSK”)forskolin treatment and the fold induction (response) due to forskolintreatment (“FOLD”).

FIG. 57 shows the luminescence generated from lysed HEK293 cellsexpressing 9B8 opt and 9B8 opt+K33N (“K33N”) cAMP transcriptionalreporters using the novel coelenterazine PBI-3939 as a substrate with(“Induced”) or without (“Basal”) forskolin treatment and the foldinduction due to forskolin treatment (“Fold Induction”).

FIGS. 58A-C show the luminescence of the OgLuc variants 9B8 and L27Vlytic reporter constructs for multiple pathways in multiple cell types.

FIGS. 59A-C show the luminescence of the OgLuc variant L27V reporterconstructs in various cell lines and with various response elements.

FIGS. 60A-B show the luminescence of the OgLuc variant L27V secretablereporter compared to Metridia longa luciferase with a CMV promoter (FIG.60A) or a NFkB response element (FIG. 60B).

FIGS. 61A-F show the absolute luminescence (FIGS. 61A and 61B), thenormalized luminescence (FIGS. 61C and 61D) and the fold response (FIGS.61E and 61F) of optimized versions of L27V (L27V01, L27V02 and L27V03)compared to L27V (L27V00) expressed in HeLa cells.

FIGS. 62A-B show the luminescence of secreted OgLuc variant L27V02(containing the IL-6 secretion signal) reporter (FIG. 62A) and L27V02(“L27V(02)”), L27V02P (“L27V(02)P(01)”) and Luc2 (“Fluc”) reporters(FIG. 62A) expressed in HepG2 cells treated with various doses of rhTNFα(“TNFα”).

FIG. 63 shows the luminescence generated from media and lysate samplesof HEK293 cells expressing the codon optimized variant IV opt with orwithout the secretion signal sequence using the novel PBI-3939 as asubstrate compared to hRL (“Renilla”) with or without the secretionsignal sequence using native coelenterazine as a substrate.

FIGS. 64A-D show the luminescence of the secreted OgLuc variants 9B8, V2and L27V reporters expressed in CHO cells (FIGS. 64A and 64B) and HeLa(FIGS. 64C and 64D).

FIGS. 65A-B show a comparison of the luminescence from the secretedOgLuc variants 9B8 and V2 using PBI-3939 as a substrate to that of thesecreted luciferase of Metridia longa using Ready-to-Glow™ as asubstrate numerically (FIG. 65A) and graphically (FIG. 65B).

FIGS. 66A-B show the fold-increase in luminescence over backgroundgenerated from HEK293 cells expressing hRL (“Ren”) and 9B8 opt using thecoelenterazine derivatives ENDUREN™ (FIG. 66A) and VIVIREN™ (FIG. 66B)and the novel coelenterazine PBI-3939 (FIG. 66B) as substrates.

FIGS. 67A-D show confocal images of U20S cells transiently expressingL27V-HaloTag® fusion (FIG. 67A) or IL6-L27V fusion (FIGS. 67B-D). Scalebars=20 μm.

FIG. 68 shows the luminescence generated from lysed bacterial cellsexpressing various OgLuc variants and hRL (“Renilla”) in the presence(“Sand”) or absence of sandwich background (“pF4Ag”) using nativecoelenterazine as a substrate.

FIG. 69 shows the fold-decrease in activity of various OgLuc variantsand hRL (“Renilla”) due to the presence of the sandwich background usingnative coelenterazine as a substrate.

FIG. 70 shows the fold-decrease in activity of 9B8 opt and 9B8 opt+K33Nin bacterial lysates due to the presence of the sandwich backgroundusing the novel coelenterazine PBI-3939 as a substrate.

FIG. 71 shows the spectral profile of the OgLuc variant L27V.

FIG. 72 shows the luminescence of two circulated permuted (CP) versionsof the OgLuc variant L27V, CP84 and CP95, either with no linker or witha 5, 10, or 20 amino acid linker.

FIGS. 73A-G show the luminescence of the various CP-TEV protease L27Vconstructs expressed in wheat germ extract (FIGS. 73A-D), E. coli (FIG.73F-G) and HEK 293 cells (FIG. 73H). FIGS. 73A-D show the basalluminescence of the various CP-TEV protease L27V constructs prior to TEVaddition. FIG. 73E shows the response of the CP-TEV protease L27Vconstructs of FIGS. 73A-D.

FIG. 74 shows the fold response of various protein complementation L27Vpairs.

FIGS. 75A-C show the luminescence of various protein complementation(PCA) L27V pairs: one L27V fragment of each pair was fused to eitherFKBP or FRB using a 1/4 configuration (FIG. 75A) or a 2/3 configuration(FIG. 75B), and the interaction of FKBP and FRB monitored in HEK 293cells. The luminescence of various protein complementation (PCA)negative controls was also monitored (FIG. 75C).

FIGS. 76A-H show the luminescence of various protein complementation(PCA) L27V pairs: one L27V fragment of each pair was fused to eitherFKBP or FRB using a 2/3 configuration (FIGS. 76A and 76C) or a 1/4configuration (FIGS. 76B and 76D), and the interaction of FKBP and FRBmonitored in wheat germ extract (FIGS. 76A and 76B) and rabbitreticulocyte lysate (RRL) (FIGS. 76C and 76D). The luminescence ofvarious protein complementation (PCA) negative controls was alsomeasured (FIG. 76E) in cell free system. The 1/4 configuration was usedin a cell free system (FIG. 76F), HEK293 cells (FIG. 76G) and in a lyticsystem (FIG. 76H).

FIGS. 77A-C show the luminescence of various protein complementationL27V pairs treated with FK506 and rapamycin (FIG. 77A) and the chemicalstructure of FK506 (FIG. 77A) and rapamycin (FIG. 77B).

FIG. 78 shows the activity of the OgLuc variant 9B8 cAMP biosensor withforskolin treatment.

FIGS. 79A-D show the luminescence of circularly permuted (FIGS. 79A and79C) and straight split (FIGS. 79B and 79D) L27V variants in rabbitreticulocyte lysate (FIGS. 79A-B) and HEK293 cells (FIGS. 79C-D).

FIGS. 80A-B show the subcellular distribution of the OgLuc variant L27V(FIG. 80A) and control vector pGEM3ZF (FIG. 80B) in U20S cells forvarious exposure times.

FIGS. 81A-C show the subcellular location of the OgLuc variant L27Vfused to either the transcription factor Nrf2 (FIG. 81B) or GPCR (FIG.81C) compared to an unfused L27V control (FIG. 81A).

FIGS. 82A-C show the use of the OgLuc variant 9B8 opt to monitorintracellular signaling pathways using PBI-4377 (FIG. 82A). The 9B8 optluciferase was fused to either IkB (FIG. 82B) or ODD (oxygen-dependentdegradation domain of Hif-1α) (FIG. 82C), and fold response to astimulus (TNFα for IkB and phenanthroline for ODD) was monitored vialuminescence.

FIGS. 83A-C show the monitoring of oxidative stress signal pathwaysusing the OgLuc variant (FIG. 83A), L27V02 (FIG. 83B), or Renillaluciferase (Rluc) (FIG. 83C).

FIGS. 84A-B show the comparison of the Nrf2-L27V02 sensor (FIG. 84A) andNrf2(ARE)-Luc2P reporter (FIG. 84B).

FIGS. 85A-B show the emission spectra of IV-HT7 with and without ligand,using 1 μM TMR (FIG. 85A) or 10 μM Rhodamine 110 (FIG. 85B) as a ligandfor HT7 and coelenterazine-h as a substrate for IV.

FIG. 86 shows the luminescence generated from lysed bacterial cellsexpressing 9B8 opt mixed with (“+ caspase”) or without (“no caspase”)caspase-3 using a pro-coelenterazine substrate.

FIGS. 87A-C show, the luminescence generated from circularly permuted,straight split L27V variants CP84 and CP103 using PBI-3939 as asubstrate with (FIG. 87B) or without (not shown) rapamycin treatment andthe response (FIG. 87C) due to rapamycin treatment. The concept of thecircularly permuted straight split variants is shown in FIG. 87A.

FIG. 88 shows percent remaining activity of the L27V variant afterexposure to various amounts of urea.

FIG. 89 shows the effect of 3M urea on the activity of the L27V variant.

FIGS. 90A-B show the bioluminescence imaging of hormone-induced nuclearreceptor (NR) translocation of OgLuc fusions using PBI-3939 substrate.

FIGS. 91A-B show the bioluminescence imaging of phorbol ester-inducedProtein Kinase C alpha (PKC alpha) translocation of OgLuc fusions usingPBI-3939 substrate.

FIGS. 92A-B show the bioluminescence imaging of autophagosomal proteintranslocation of OgLuc fusions using PBI-3939 substrate.

DETAILED DESCRIPTION

Before any embodiments of the invention are explained in detail, it isto be understood that the invention is not limited in its application tothe details of structure, synthesis, and arrangement of components setforth in the following description or illustrated in the followingdrawings. The invention is described with respect to specificembodiments and techniques, however, the invention is capable of otherembodiments and of being practiced or of being carried out in variousways.

In the following description of the methods of the invention, processsteps are carried out at room temperature (about 22° C.) and atmosphericpressure unless otherwise specified. It also is specifically understoodthat any numerical range recited herein includes all values from thelower value to the upper value. For example, if a concentration range orbeneficial effect range is stated as 1% to 50%, it is intended thatvalues such as 2% to 40%, 10% to 30%, or 1% to 3%, etc. are expresslyenumerated in this specification. Similarly, if a sequence identityrange is given as between, e.g., 60% to <100%, it is intended thatintermediate values such as 65%, 75%, 85%, 90%, 95%, etc. are expresslyenumerated in this specification. These are only examples of what isspecifically intended, and all possible numerical values from the lowestvalue to the highest value are considered expressly stated in theapplication.

Unless expressly specified otherwise, the term “comprising” is used inthe context of the present application to indicate that further membersmay optionally be present in addition to the members of the listintroduced by “comprising”. It is, however, contemplated as a specificembodiment of the present invention that the term “comprising”encompasses the possibility of no further members being present, i.e.,for the purpose of this embodiment “comprising” is to be understood ashaving the meaning of “consisting of”.

The following detailed description discloses specific and/or preferredvariants of the individual features of the invention. The presentinvention also contemplates, as particularly preferred embodiments,those embodiments which are generated by combining two or more of thespecific and/or preferred variants described for two or more of thefeatures of the present invention.

Unless expressly specified otherwise, all indications of relativeamounts in the present application are made on a weight/weight basis.Indications of relative amounts of a component characterized by ageneric term are meant to refer to the total amount of all specificvariants or members covered by said generic term. If a certain componentdefined by a generic term is specified to be present in a certainrelative amount, and if this component is further characterized to be aspecific variant or member covered by the generic term, it is meant thatno other variants or members covered by the generic term areadditionally present such that the total relative amount of componentscovered by the generic term exceeds the specified relative amount. Morepreferably, no other variants or members covered by the generic term arepresent at all.

Overview

In various aspects, the invention is drawn to novel compounds, novelluciferases, and combinations thereof. The invention encompassesmethods, compositions, and kits including the novel compounds, novelluciferases, and/or combinations thereof.

The novel compounds are novel coelenterazines, which can be used assubstrates by proteins that utilize coelenterazines to produceluminescence, including, but not limited to, luciferases andphotoproteins found in various marine organisms such as cnidarians(e.g., Renilla luciferase), jellyfish (e.g., aequorin from the Aequoreajellyfish) and decapods luciferases (e.g., luciferase complex ofOplophorus gracilirostris). In various embodiments, the novelcoelenterazines of the present invention have at least one of enhancedphysical stability (e.g., enhanced coelenterazine stability), reducedautoluminescence, and increased biocompatibility with cells (e.g., lesstoxic to cells, including heterologous cell types) relative to nativecoelenterazine.

The novel luciferases disclosed herein include variants of the activesubunit of a decapod luciferase. The novel luciferases can utilizevarious coelenterazines as substrates, including, but not limited to,native and known coelenterazines as well as the novel coelenterazines ofthe present invention. The novel luciferases display at least one of:enhanced luminescence (including increased brightness, enhanced signalstability and/or signal duration); enhanced enzyme stability (i.e.,enhanced enzymatic activity including enhanced resistance to elevatedtemperature, changes in pH, inhibitors, denaturants, and/or detergents);altered substrate specificity (i.e., change in relative substratespecificity); and enhanced biocompatibility (including at least one ofimproved expression in cells, reduced toxicity, and/or cell stress). Invarious embodiments, the present invention encompasses novel luciferasesthat are present in solution as soluble, active monomers, chemicallylinked to other molecules (e.g., fusion proteins), or attached onto asolid surface (e.g., particles, capillaries, or assay tubes or plates).

Certain combinations of the novel coelenterazines and the novelluciferases provide significant technical advantages for bioluminescentassays including enhanced luminescence, wherein enhanced luminescencemay be due to one or more factors including enhanced signal stabilityand enhanced coelenterazine stability. Additionally, many of the novelcoelenterazines were designed to be smaller than commercially-availableand/or known coelenterazines. In some cases, the novel luciferases ofthe present invention preferentially utilize the novel, smallercoelenterazines over the commercially-available and/or known largercoelenterazines.

The invention encompasses combinations of: the novel luciferase variantswith the novel coelenterazines; the novel luciferase variants with knownor native coelenterazines; and the novel coelenterazines with any knownor native protein (e.g., luciferases or photoproteins) that usescoelenterazine as a substrate.

The term “coelenterazine” refers to naturally-occurring (“native”)coelenterazine as well as analogs thereof, including coelenterazine-n,coelenterazine-f, coelenterazine-h, coelenterazine-hcp,coelenterazine-cp, coelenterazine-c, coelenterazine-e,coelenterazine-fcp, bis-deoxycoelenterazine (“coelenterazine-hh”),coelenterazine-i, coelenterazine-icp, coelenterazine-v, and 2-methylcoelenterazine, in addition to those disclosed in WO 2003/040100 andU.S. application Ser. No. 12/056,073 (paragraph [0086]), the disclosuresof which are incorporated by reference herein. The term “coelenterazine”also refers to the novel coelenterazines disclosed herein (see below).The term “known coelenterazine” refers to a coelenterazine analog knownprior to the present invention.

The term “OgLuc” refers to a decapod luciferase protein, or a variant ofsuch a protein, which generates light in the presence of acoelenterazine. The OgLuc protein may, in its naturally-occurring form,be a monomer or may be a subunit of a protein complex. The OgLuc used inthe exemplary embodiments disclosed herein is the 19 kDa subunit fromthe luciferase complex of Oplophorus gracilirostris, although comparablepolypeptides from other decapod species (including other Oplophorusspecies) could also be employed and are encompassed within the invention(see R. D. Dennell, Observations on the luminescence of bathypelagicCrustacea decapoda of the Bermuda area, Zool. J Linn. Soc., Lond. 42(1955), pp. 393-406; see also Poupin et al. September 1999. Inventairedocumenté des espècies et bilan des formes les plus communes de la merd'Iroise. Rapport Scientifique du Laboratoire d'Océanographie de l'ÉcoleNavale (LOEN), Brest (83 pgs), each of which is incorporated byreference herein); examples include, without limitation, luciferases ofthe Aristeidae family, including Plesiopenaeus coruscans; the Pandalideafamily, including Heterocarpus and Parapandalus richardi, theSolenoceridae family, including Hymenopenaeus debilis and Mesopenaeustropicalis; the Luciferidae family, including Lucifer typus; theSergestidae family, including Sergestes atlanticus, Sergestes arcticus,Sergestes armatus, Sergestes pediformis, Sergestes cornutus, Sergestesedwardsi, Sergestes henseni, Sergestes pectinatus, Sergestes sargassi,Sergestes similis, Sergestes vigilax, Sergia challengeri, Sergiagrandis, Sergia lucens, Sergia prehensilis, Sergia potens, Sergiarobusta, Sergia scintillans, and Sergia splendens; the Pasiphaeidaefamily, including Glyphus marsupialis, Leptochela bermudensis,Parapasiphae sulcatifrons, and Pasiphea tarda; the Oplophoridae family,including Acanthephyra acanthitelsonis, Acanthephyra acutifrons,Acanthephyra brevirostris, Acanthephyra cucullata, Acanthephyracurtirostris, Acanthephyra eximia, Acanthephyra gracilipes, Acanthephyrakingsleyi, Acanthephyra media, Acanthephyra microphthalma, Acanthephyrapelagica, Acanthephyra prionota, Acanthephyra purpurea, Acanthephyrasanguinea, Acanthephyra sibogae, Acanthephyra stylorostratis, Ephyrinabifida, Ephyrina figueirai, Ephyrina koskynii, Ephyrina ombango,Hymenodora glacialis, Hymenodora gracilis, Meningodora miccyla,Meningodora mollis, Meningodora vesca, Notostomus gibbosus, Notostomusauriculatus, Oplophorus gracilirostris, Oplophorus grimaldii, Oplophorusnovaezealandiae, Oplophorus spinicauda, Oplophorus foliaceus, Oplophorusspinosus, Oplophorus typus, Systellaspis braueri, Systellaspis cristata,Systellaspis debilis, and Systellaspis pellucida; and theThalassocaridae family, including Chlorotocoides spinicauda,Thalassocaris crinita, and Thalassocaris lucida.

The polypeptide sequence of the mature (i.e., with no signal sequence)19 kDa subunit of the naturally-occurring form of the Oplophorusgracilirostris luciferase (i.e., 169 amino acids, residues 28 to 196 ofBAB 13776) is given in SEQ ID NO: 1. In various embodiments, amethionine residue and a valine residue are inserted at the beginning ofthe synthetic OgLuc sequence (e.g., as indicated in the C1+A4Epolypeptide sequence, SEQ ID NO: 3) to facilitate cloning and expressionin heterologous systems. Nevertheless, for consistency, the positionnumbers of the various amino acid substitutions referred to herein arespecified “relative to” SEQ ID NO: 1, i.e., the polypeptide sequence ofthe mature (with no signal sequence), native 19 kDa subunit of theOplophorus gracilirostris luciferase protein complex.

Specifically, a protein is a decapod luciferase if, upon alignment ofits amino acid sequence with SEQ ID NO: 1, the sequence identityis >30%, preferably >40%, and most preferably >50%, and the protein canutilize coelenterazine as a substrate to catalyze the emission ofluminescence, and the amino acid sequence beginning at the positioncorresponding to position 8 of SEQ ID NO: 1 is:

(VII) (SEQ ID NO. 330)[GSAIVK]-{FE}-[FYW]-x-[LIVMFSYQ]-x-x-{K}-x-[NHGK]-x-[DE]-x-[LIVMFY]-[LIVMWF]-x-{G}-[LIVMAKRG],

with no more than 5 differences, or more preferably no more than 4, 3,2, or 1 difference, or most preferably no differences, wherein thedifferences occur in positions corresponding to pattern position 1, 2,3, 5, 8, 10, 12, 14, 15, 17, or 18 of Formula (VII) according to Table4. Differences may also include gaps or insertions between the patternpositions of Table 4.

The term “variant” refers to a modified version of a startingpolypeptide or polynucleotide sequence. The term “parental” is arelative term that refers to a starting sequence which is then modified.The parental sequence is generally used as a reference for the proteinencoded by the resulting modified sequence, e.g., to compare theactivity levels or other properties of the proteins encoded by theparental and the modified sequences. The starting sequence can be anaturally-occurring (i.e., native or wild-type) sequence. The startingsequence can also be a variant sequence which is then further modified.A polypeptide sequence is “modified” when one or more amino acids (whichmay be naturally-occurring or synthetic) are substituted, deleted,and/or added at the beginning, middle, and/or end of the sequence. Apolynucleotide sequence is “modified” when one or more nucleotides aresubstituted, deleted, and/or added at the beginning, middle, and/or endof the sequence, but which may or may not alter the amino acid encodedby the sequence. In some embodiments, the modifications produce avariant that is a functional fragment of a particular OgLuc or OgLucvariant. A functional fragment is a fragment which is less than afull-length parental sequence which has the same functional activity asthe full-length parental sequence. Functional activity is the ability toexhibit luminescence. In some embodiments, the modifications produce avariant that is a permuted sequence of the parental sequence, such as acircularly permuted sequence and permuted sequences comprising deletionsand/or insertions.

Several of the OgLuc variants disclosed herein have been assignedshorthand names to facilitate discussion. The term “C1+A4E” (alsoreferred to as “C1A4E”) refers to a particular OgLuc variant with theamino acid substitutions A4E, Q11R, A33K, V44I, A54F, P115E, Q124K,Y138I, and N166R relative to SEQ ID NO: 1 (SEQ ID NOs: 2 and 3) (wherethe format “x#y” indicates a parent amino acid ‘x’ at a position ‘#’that is changed to variant amino acid ‘y’). Variants of the C1+A4E OgLucvariant which are presented herein contain at least the amino acidsubstitutions found in C1+A4E, unless otherwise indicated. The term“IVY” refers to a variant of the C1+A4E OgLuc variant having additionalamino acid substitutions F54I, I90V, and F77Y relative to SEQ ID NO: 1(SEQ ID NOs: 8 and 9). The term “IV” refers to another variant of theC1+A4E OgLuc variant having additional amino acid substitutions F541 and190V relative to SEQ ID NO: 1 (SEQ ID NOs: 14 and 15). The term “QC27”refers to yet another variant of the C1+A4E OgLuc variant havingadditional amino acid substitutions Q18L, F54I, L92H, and Y109F relativeto SEQ ID NO: 1 (SEQ ID NOs: 4 and 5). The term “QC27-9a” refers to avariant of the QC27 OgLuc variant with additional amino acidsubstitutions V21L, F68Y, L72Q, M75K, H92R, and V158F relative to SEQ IDNO: 1 (SEQ ID NOs: 6 and 7). The term “9B8” refers to a variant of theIV OgLuc variant with additional amino acid substitutions Q18L, F68Y,L72Q, and M75K relative to SEQ ID NO: 1 (SEQ ID NOs: 18 and 19). Theterm “9B8 opt” refers to the codon optimized version of the 9B8 variant(SEQ ID NO: 24). The term “9B8 opt+K33N” refers to a variant of the 9B8opt variant with additional amino acid substitution K33N relative to SEQID NO: 1 (SEQ ID NOs: 42 and 43). The term “9B8 opt+K33N+170G” refers toa variant of the “9B8 opt+K33N” variant with an additional glycineappended to the C-terminus of the variant, i.e., 170G relative to SEQ IDNO: 1 (SEQ ID NO: 68 and 69). The terms “L27V+T39T+K43R+Y68D” and “L27V”refers to a variant of the 9B8 opt+K33N″ variant with additional aminoacid substitutions L27V, T39T, K43R, and Y68D relative to SEQ ID NO: 1(SEQ ID NOs: 88 and 89). The terms “T39T+K43R+Y68D” and “V2” refers to avariant of the “9B8 opt+K33N” variant with additional amino acidsubstitutions T39T, K43R, and Y68D relative to SEQ ID NO: 1 (SEQ ID NOs:92 and 93).

In general, “enhanced” means that the particular property (e.g.,luminescence, signal stability, biocompatibility, protein stability(e.g., enzyme stability), or protein expression) is increased relativeto that of the reference luciferase plus coelenterazine combination orluciferase under consideration, where the increase is at least 1%, atleast 5%, at least 10%, at least 20%, at least 25%, at least 50%, atleast 75%, at least 90%, at least 100%, at least 200%, at least 500%, orat least 1000% greater than the reference luciferase plus coelenterazinecombination or luciferase under consideration.

The term “luminescence” refers to the light output of the OgLuc variantunder appropriate conditions, e.g., in the presence of a suitablesubstrate such as a coelenterazine. The light output may be measured asan instantaneous or near-instantaneous measure of light output (which issometimes referred to as “T=0” luminescence or “flash”) at the start ofthe luminescence reaction, which may be initiated upon addition of thecoelenterazine substrate. The luminescence reaction in variousembodiments is carried out in a solution. In other embodiments, theluminescence reaction is carried out on a solid support. The solutionmay contain a lysate, for example from the cells in a prokaryotic oreukaryotic expression system. In other embodiments, expression occurs ina cell-free system, or the luciferase protein is secreted into anextracellular medium, such that, in the latter case, it is not necessaryto produce a lysate. In some embodiments, the reaction is started byinjecting appropriate materials, e.g., coelenterazine, buffer, etc.,into a reaction chamber (e.g., a well of a multiwell plate such as a96-well plate) containing the luminescent protein. In still otherembodiments, the OgLuc variant and/or novel coelenterazine areintroduced into a host and measurements of luminescence are made on thehost or a portion thereof, which can include a whole organism or cells,tissues, explants, or extracts thereof. The reaction chamber may besituated in a reading device which can measure the light output, e.g.,using a luminometer or photomultiplier. The light output or luminescencemay also be measured over time, for example in the same reaction chamberfor a period of seconds, minutes, hours, etc. The light output orluminescence may be reported as the average over time, the half-life ofdecay of signal, the sum of the signal over a period of time, or thepeak output. Luminescence may be measured in Relative Light Units(RLUs).

The “enhanced luminescence” of an OgLuc variant may be due to one ormore of the following characteristics: enhanced light output (i.e.,brightness), enhanced substrate specificity, enhanced signal stability,and/or enhanced signal duration. Enhanced signal stability includes anincrease in how long the signal from a luciferase continues toluminesce, for example, as measured by the half-life of decay of thesignal in a time-course. Enhanced luminescence may be determinedrelative to the comparable property of a luciferase such as wild-typeOgLuc, an OgLuc variant protein, Renilla luciferase (e.g., hRluc), orfirefly luciferase (e.g., Luc2 luciferase from Photinus pyralis)combined with a native, known, or novel substrate, as shown in theExamples below. For example, the luminescence of a given OgLuc variantin combination with a particular coelenterazine (including native,known, or novel coelenterazines) may be compared to the properties ofone of OgLuc variants C1+A4E, IV, or IVY combined with any of a native,known, or novel coelenterazine disclosed herein, using one or more ofthe assays disclosed in the Examples below. In particular, enhancedluminescence may be determined by measuring the luminescence signal(RLU) resulting from the incubation of bacterial lysates containingOgLuc variants in question with the substrate, PBI-3939. Measurementsare taken in a reagent which may contain TERGITOL™ to provide Glo-likekinetics, e.g., in which enzyme inactivation is slowed and theluminescence signal is stabilized, which is described elsewhere in theapplication. In some embodiments, some luciferase variants, e.g., L27V,with certain compounds, e.g., PRE-3939, provide extended duration of theluminescent emission, or glow-like kinetics, in the absence ofTERGITOL™. The luminescence signal may be compared to that of areference point such as the C1+A4E variant with coelenterazine orcoelenterazine-h or Renilla luciferase with native coelenterazine.

“Enzyme stability” refers to the stability of enzyme activity (i.e.,tolerance of enzymatic activity to reaction conditions). Enhanced enzymestability refers to enhanced stability of enzyme activity (i.e.,enhanced tolerance to reaction conditions). Enhanced enzyme stabilityincludes enhanced thermal stability (e.g., stability at elevatedtemperatures) and chemical stability (e.g., stability in the presence ofinhibitors or denaturants such as detergents, including, e.g., TRITON™X-100). Enzyme stability can be used as a measure of protein stability,particularly under conditions known to be disruptive of proteinstructure, such as high temperatures or the presence of chemicaldenaturants. In particular, enhanced protein stability may be determinedusing thermal analysis as described elsewhere in the application (e.g.,in Example 28). The luminescence signal may be compared to the referencepoint of C1+A4E variant with coelenterazine or coelenterazine-h orRenilla luciferase with native coelenterazine.

“Biocompatibility” refers to the tolerance of a cell (e.g., prokaryoticor eukaryotic) to a luciferase and/or coelenterazine compound.Biocompatibility of a luciferase and/or coelenterazine compound isrelated to the stress it causes on the host cell. For example, aluciferase that is not tolerated by the cell (i.e., one that stresses acell) may not be expressed efficiently within the cell, for example, theluciferase may be expressed within the cell, but exhibit reducedactivity due to the formation of inclusion bodies by the expressedprotein. Biocompatibility of a luciferase is related to the ability ofthe cells to tolerate the insertion of the foreign gene, i.e., atransgene containing the gene encoding the luciferase or fragmentthereof, whereby the cells with the transgene do not exhibitmanifestations of stress, including induction of stress responsepathways, reduced rate of growth, and/or reduced viability (e.g.,reduced number of living cells, reduced membrane integrity, or increasedrates of apoptosis). Other indications of cell stress may includechanges in gene expression, signaling pathways, and/or regulatorypathways. Enhanced biocompatibility of an OgLuc variant may be due tofactors such as enhanced protein expression and/or reduced cell stress.Enhanced expression of luminescence for a particular polynucleotideencoding an OgLuc variant may be determined relative to luminescenceexpression levels for a polynucleotide encoding wild-type OgLuc or anOgLuc variant protein, including codon-optimized polynucleotides, whereluminescence activity can be used as a means to monitor proteinexpression levels.

In particular, enhanced biocompatibility of the OgLuc variant, novelcoelenterazine compound and/or a combination thereof, may be determinedby measuring cell viability and/or growth rate of cells. For example,enhanced biocompatibility of the OgLuc variants may be determined bymeasuring cell viability and/or growth rate of cells containing theOgLuc variants compared to cells containing firefly or Renillaluciferase or no luciferase, in the absence of any coelenterazinecompound to determine how compatible and/or toxic the luciferase is tothe cells. Enhanced biocompatibility of the novel coelenterazinecompounds may be determined by measuring cell viability in the absenceof luciferase expression of cells exposed to the novel coelenterazinecompound compared to native or known coelenterazines to determine howcompatible and/or toxic the coelenterazine compound is to the cells.Enhanced biocompatibility of a combination of an OgLuc variant with anovel coelenterazine compound may be determined by measuring cellviability and/or growth rate of cells containing the OgLuc variant andexposed to the novel coelenterazine and compared to cells containingfirefly or Renilla luciferase or no luciferase and exposed to native orknown coelenterazines.

In particular, enhanced biocompatibility may be determined using cellviability analysis as described elsewhere in the application (e.g.,using a CELLTITER-GLO® assay as described in Example 18 or an apoptosisassay such as one using CASPASE-GLO® technology according to themanufacturer's instructions) or one known in the art. The effect of anOgLuc variant on cell viability or apoptosis may be compared to theeffect of a reference luciferase, such as the C1+A4E variant, a fireflyluciferase or Renilla luciferase. The effect of the novel coelenterazinecompound on cell viability or apoptosis may be compared to the effect ofnative or known coelenterazine compounds on cell viability or apoptosis.

Enhanced biocompatibility may also be determined by measuring the effectof the OgLuc variant and/or novel coelenterazine compound on cell growthor gene expression. For examples, enhanced biocompatibility of the OgLucvariant may be determined by measuring the cell number after a period oftime or by determining the expression of stress response genes in asample of cells that contain the OgLuc variant compared to cells thatcontain another luciferase or no luciferase. Enhanced biocompatibilityof the novel coelenterazine compound may be determined by measuring thecell number after a period of time or by determining the expression ofstress response genes in a sample of cells that are exposed to the novelcoelenterazine compound compared to cells exposed to native or knowncoelenterazines or no coelenterazines. The effect of the OgLuc varianton cell growth or gene expression may be compared to a referenceluciferase, such as C1+A4E variant, a firefly luciferase or Renillaluciferase. The effect of the novel coelenterazine on cell growth orgene expression may be compared to native or known coelenterazines.

The identification of robust, stable cell lines expressing an OgLucvariant of the present invention, either in the cytoplasm or as asecreted form, can be facilitated by the bright signal of the luciferaseand the small size of the OgLuc gene. The relatively small gene sequenceis expected to reduce the likelihood of genetic instability resultingfrom the integration of the foreign DNA into a cell's genome. As aresult of the increased brightness of the OgLuc variants and/or thenovel coelenterazines of the present invention, less protein expression,and thereby less DNA needed for transfection, may produce a given levelof brightness compared to other known luciferases such as native OgLuc,firefly, or Renilla luciferase, which contributes to an enhancedbiocompatibility for the OgLuc variants and/or novel coelenterazines.Enhanced biocompatibility of the OgLuc variants may be measured by theamount of DNA or reagents, e.g., transfection chemicals, needed intransient transfections to generate cells with the same level ofluminescence as cells transfected with other luciferases, e.g., nativeOgLuc, firefly or Renilla luciferase. In some embodiments, the amount ofOgLuc variant DNA or reagents needed for transfection is less than theamount needed for another luciferase, e.g., native OgLuc, firefly, orRenilla luciferase, to generate transfected cells with the same level ofluminescence obtained with the other luciferase. Enhancedbiocompatibility of the OgLuc variants may be measured by the recoverytime of the cells after transfection. In some embodiments, the amount oftime needed for recovery after transfection with the OgLuc variant isless than the time needed for another luciferase, e.g., native OgLuc,firefly or Renilla luciferase.

“Relative substrate specificity” is determined by dividing theluminescence of a luciferase in the presence of a test coelenterazinesubstrate by the luminescence of the luciferase in the presence of areference coelenterazine substrate. For example, relative specificitymay be determined by dividing the luminescence of a luciferase with anovel coelenterazine of the present invention by the luminescence of theluciferase with a different coelenterazine (e.g., native or knowncoelenterazine, see FIG. 1 for examples, or a different novelcoelenterazine of the present invention). The test coelenterazinesubstrate and the reference coelenterazine substrate that are comparedare considered a comparison substrate pair for determining relativesubstrate specificity.

A “change in relative substrate specificity” is determined by dividingthe relative substrate specificity of a test luciferase using acomparison substrate pair by the relative substrate specificity of areference luciferase using the same comparison substrate pair. Forexample, a change in relative specificity may be determined by dividingthe relative substrate specificity of a test luciferase with a novelcoelenterazine of the present invention compared to a differentcoelenterazine (e.g., native or known coelenterazine or a differentnovel coelenterazine of the present invention), by the relativesubstrate specificity of a reference luciferase with the same novelcoelenterazine of the present invention compared to the same differentcoelenterazine used for the test luciferase.

In some embodiments, the luminescence with one novel coelenterazine iscompared to the luminescence with a different novel coelenterazine. Insome embodiments, the luminescence with one native or knowncoelenterazine is compared to the luminescence with another native orknown coelenterazine. In still other embodiments, the luminescence withone native or known coelenterazine is compared to the luminescence witha novel coelenterazine.

The novel coelenterazines of the present invention include propertiessuch as enhanced physical stability (e.g., enhanced coelenterazinestability) or reduced autoluminescence. The physical stability of thecoelenterazine refers to how stable the coelenterazine is in certainconditions such that it maintains the ability to luminesce when used asa substrate by a luciferase. Luminescence that is not dependent on theactivity of a luciferase or photoprotein is termed autoluminescence.Autoluminescence is the luminescence of a substance produced by energyreleased in the form of light during decay or decomposition. Forexample, autoluminescence can be caused by spontaneous oxidation of theluminogenic substrate coelenterazine.

As used herein, “pure” or “purified” means an object species is thepredominant species present (i.e., on a molar and/or mass basis, it ismore abundant than any other individual species, apart from water,solvents, buffers, or other common components of an aqueous system inthe composition), and, in some embodiments, a purified fraction is acomposition wherein the object species comprises at least about 50% (ona molar basis) of all macromolecular species present. Generally, a“substantially pure” composition will comprise more than about 80% ofall macromolecular species present in the composition, in someembodiments more than about 85%, more than about 90%, more than about95%, or more than about 99%. In some embodiments, the object species ispurified to essential homogeneity (contaminant species cannot bedetected in the composition by conventional detection methods) whereinthe composition consists essentially of a single macromolecular species.

Coelenterazine Derivatives

In some embodiments, the present invention provides novel coelenterazinederivatives of formula (Ia) or (Ib):

-   -   wherein R² is selected from the group consisting of

or C₂₋₅ straight chain alkyl;

-   -   R⁶ is selected from the group consisting of —H, —OH, —NH₂,        —OC(O)R or —OCH₂OC(O)R;    -   R⁸ is selected from the group consisting of

H or lower cycloalkyl;

-   -   wherein R³ and R⁴ are both H or both C₁₋₂ alkyl;    -   W is —NH₂, halo, —OH, —NHC(O)R, —CO₂R;    -   X is —S—, —O— or —NR²²—;    -   Y is —H, —OH, or —OR¹¹    -   Z is —CH— or —N—;    -   each R¹¹ is independently —C(O)R″ or —CH₂OC(O)R″;    -   R²² is H, CH₃, or CH₂CH₃    -   each R is independently C₁₋₇ straight-chain alkyl or C₁₋₇        branched alkyl;    -   R″ is C₁₋₇ straight-chain alkyl or C₁₋₇ branched alkyl;    -   the dashed bonds indicate the presence of an optional ring,        which may be saturated or unsaturated;    -   with the proviso that when R² is

or

R⁸ is not

with the proviso that when R² is

R⁸ is

or lower cycloalkyl; and

-   -   with the proviso that when R⁶ is NH₂, R² is

or C₂₋₅ alkyl;

-   -   or R⁸ is not

The term “alkyl”, as used herein, pertains to a monovalent moietyobtained by removing a hydrogen atom from a hydrocarbon compound, andwhich may be saturated, partially unsaturated, or fully unsaturated. Thealkyl group may be a straight-chain or branched. An alkyl group may beoptionally substituted with, for example, halo. Examples ofstraight-chain alkyl groups include, but are not limited to, ethyl,n-propyl, n-butyl, and n-propyl, n-hexyl and n-heptyl. Examples ofunsaturated alkyl groups which have one or more carbon-carbon doublebonds include, but are not limited to, ethenyl (vinyl, —CH═CH₂),2-propenyl (allyl, —CH—CH═CH₂), and butenyl. Examples of unsaturatedalkyl which have one or more carbon-carbon triple bonds include, but arenot limited to, ethynyl and 2-propynyl (propargyl). Examples of branchedalkyl groups included isopropyl, iso-butyl, sec-butyl, t-butyl andiso-pentyl.

The term “lower cycloalkyl”, as used herein, pertains to a monovalentmoiety obtained by removing a hydrogen atom from a hydrocarbon compoundhaving from 3 to 5 carbon atoms. Examples of saturated lower cycloalkylgroups include, but are not limited to, groups such as cyclopropyl,cyclobutyl and cyclopentyl. Examples of unsaturated lower cycloalkylgroups which have one or more carbon-carbon double bonds include, butare not limited to, groups such as cyclopropenyl, cyclobutenyl andcyclopentenyl.

The term “halo”, as used herein, pertains to a halogen, such as Cl, F,Br or I.

In some embodiments, R² is

and X is O or S. In other embodiments, R² is C₂₋₅ straight chain alkyl.In certain embodiments, R⁸ is

lower cycloalkyl or H. In other embodiments, R⁸ is benzyl. In someembodiments, R″ is —C(CH₃)₃, —CH(CH₃)₂, —CH₂C(CH₃)₃, or —CH₂CH(CH₃)₂.

In some embodiments, the present invention provides compounds accordingto Formula (IIa) or (IIb):

wherein X is O or S, R⁶ is H or OH, R¹¹ is as defined above, and thedashed bonds indicate the presence of an optional ring.

In some embodiments, the invention provides compounds according toFormula (Ma) or (IIIb):

wherein R¹² is C₂₋₅ straight-chain alkyl, furyl or thienyl, R⁶ is H orOH, R¹¹ is as defined above, and the dashed bonds indicate the presenceof an optional ring.

In some embodiments, the invention provides compounds according toFormula (IVa) or (IVb):

wherein X is O or S, R⁶ is H or OH, R¹⁸ is H,

or lower cycloalkyl, R³, R⁴ and R¹¹ are as defined above, and the dashedbonds indicate the presence of an optional ring.

In some embodiments, the invention provides a compound according toFormula (Va) or (Vb):

wherein R⁸ is benzyl, R¹¹ is as defined above, and the dashed bondsindicate the presence of an optional ring.

In some embodiments, the present invention provides novel coelenterazinederivatives of formula (VIa) or (VIb):

wherein R² is selected from the group consisting of

or C₂₋₅ straight chain alkyl;

-   -   R⁶ is selected from the group consisting of —H, —OH, —NH₂,        —OC(O)R or —OCH₂OC(O)R;    -   R⁸ is selected from the group consisting of

H or lower cycloalkyl;

-   -   wherein R³ and R⁴ are both H or both C₁₋₂ alkyl;    -   W is —NH₂, halo, —OH, —NHC(O)R, —CO₂R;    -   X is —S—, —O— or —NH—;    -   Y is —H, —OH, or —OR¹¹;    -   Z is —CH— or —N—;    -   each R¹¹ is independently —C(O)R″ or —CH₂OC(O)R″;    -   each R is independently C₁₋₇ straight-chain alkyl or C₁₋₇        branched alkyl;    -   R″ is C₁₋₇ straight-chain alkyl or C₁₋₇ branched alkyl;    -   the dashed bonds indicate the presence of an optional ring,        which may be saturated or unsaturated;    -   with the proviso that when R² is

or

R⁸ is not

with the proviso that when R² is

R⁸ is

or lower cycloalkyl; and

-   -   with the proviso that when R⁶ is NH₂, R² is

or C₂₋₅ alkyl;

or R⁸ is not

Suitable compounds according to the present invention include

Isomers, Salts and Protected Forms

Certain compounds may exist in one or more particular geometric,optical, enantiomeric, diasteriomeric, epimeric, stereoisomeric,tautomeric, conformational, or anomeric forms, including but not limitedto, cis- and trans-forms; E- and Z-forms; c-, t-, and r-forms; endo andexo-forms; R-, S-, and meso-forms; D- and L-forms; d- and l-forms; (+)and (−) forms; keto-, enol-, and enolate-forms; syn- and anti-forms;synclinal- and anticlinal-forms; α- and β-forms; axial and equatorialforms; boat-, chair-, twist-, envelope-, and half-chair forms; andcombinations thereof, hereinafter collectively referred to as “isomers”(or “isomeric forms”).

Note that, except as discussed below for tautomeric forms, specificallyexcluded from the term “isomers”, as used herein, are structural (orconstitutional) isomers (i.e., isomers which differ in the connectionsbetween atoms rather than merely by the position of atoms in space). Forexample, a reference to a methoxy group, —OCH₃, is not to be construedas a reference to its structural isomer, a hydroxymethyl group, —CH₂OH.Similarly, a reference to ortho-chlorophenyl is not to be construed as areference to its structural isomer, meta-chlorophenyl. However, areference to a class of structures may well include structurallyisomeric forms falling within that class (e.g., C₁₋₇ alkyl includesn-propyl and iso-propyl; butyl includes n-, iso-, sec-, and tert-butyl;methoxyphenyl includes ortho-, meta-, and paramethoxyphenyl).

Note that specifically included in the term “isomer” are compounds withone or more isotopic substitutions. For example, H may be in anyisotopic form, including ¹H, ²H (D), and ³H (T); C may be in anyisotopic form, including ¹²C, ¹³C, and ¹⁴C; O may be in any isotopicform, including ¹⁶O and ¹⁸O; and the like.

Unless otherwise specified, a reference to a particular compoundincludes all such isomeric forms, including (wholly or partially)racemic and other mixtures thereof. Methods for the preparation (e.g.,asymmetric synthesis) and separation (e.g., fractional crystallizationand chromatographic means) of such isomeric forms are either known inthe art or are readily obtained by adapting the methods taught herein,or known methods, in a known manner.

Unless otherwise specified, a reference to a particular compound alsoincludes ionic, salt, solvate, and protected forms of thereof, forexample, as discussed below. It may be convenient or desirable toprepare, purify, and/or handle a corresponding salt of the activecompound, for example, a pharmaceutically-acceptable salt. Examples ofpharmaceutically acceptable salts are discussed in Berge et al., J.Pharm. Sci., 66:1-19 (1977).

For example, if the compound is anionic, or has a functional group whichmay be anionic (e.g., —COOH may be —COO—), then a salt may be formedwith a suitable cation. Examples of suitable inorganic cations include,but are not limited to, alkali metal ions such as Na⁺ and K⁺, alkalineearth cations such as Ca²⁺ and Mg²⁺, and other cations such as Al³⁺.Examples of suitable organic cations include, but are not limited to,ammonium ion (i.e., NH⁴⁺) and substituted ammonium ions (e.g., NH₃R⁺,NH₂R₂ ⁺, NHR₃ ⁺, NR₄ ⁺). Examples of some suitable substituted ammoniumions are those derived from: ethylamine, diethylamine,dicyclohexylamine, triethylamine, butylamine, ethylenediamine,ethanolamine, diethanolamine, piperazine, benzylamine,phenylbenzylamine, choline, meglumine, and tromethamine, as well asamino acids, such as lysine and arginine. An example of a commonquaternary ammonium ion is N(CH₃)₄ ⁺.

If the compound is cationic, or has a functional group which may becationic (e.g., —NH₂ may be —NH₃), then a salt may be formed with asuitable anion. Examples of suitable inorganic anions include, but arenot limited to, those derived from the following inorganic acids:hydrochloric, hydrobromic, hydroiodic, sulfuric, sulfurous, nitric,nitrous, phosphoric, and phosphorous. Examples of suitable organicanions include, but are not limited to, those derived from the followingorganic acids: acetic, propionic, succinic, glycolic, stearic, palmitic,lactic, malic, pamoic, tartaric, citric, gluconic, ascorbic, maleic,hydroxymaleic, phenylacetic, glutamic, aspartic, benzoic, cinnamic,pyruvic, salicyclic, sulfanilic, 2-acetyoxybenzoic, fumaric,phenylsulfonic, toluenesulfonic, methanesulfonic, ethanesulfonic, ethanedisulfonic, oxalic, pantothenic, isethionic, valeric, lactobionic, andgluconic. Examples of suitable polymeric anions include, but are notlimited to, those derived from the following polymeric acids: tannicacid, carboxymethyl cellulose.

It may be convenient or desirable to prepare, purify, and/or handle acorresponding solvate of the active compound. The term “solvate” is usedherein in the conventional sense to refer to a complex of solute (e.g.,active compound, salt of active compound) and solvent. If the solvent iswater, the solvate may be conveniently referred to as a hydrate, forexample, a mono-hydrate, a di-hydrate, a tri-hydrate, etc.

It may be convenient or desirable to prepare, purify, and/or handle theactive compound in a chemically protected form. The term “chemicallyprotected form”, as used herein, pertains to a compound in which one ormore reactive functional groups are protected from undesirable chemicalreactions, that is, are in the form of a protected or protecting group(also known as a masked or masking group or a blocked or blockinggroup). By protecting a reactive functional group, reactions involvingother unprotected reactive functional groups can be performed, withoutaffecting the protected group; the protecting group may be removed,usually in a subsequent step, without substantially affecting theremainder of the molecule. See, for example, Protective Groups inOrganic Synthesis (T. Green and P. Wuts, Wiley, 1999).

For example, a hydroxy group may be protected as an ether (—OR) or anester (—OC(═O)R), for example, as: a t-butyl ether; a benzyl, benzhydryl(diphenylmethyl), or trityl (triphenylmethyl)ether; a trimethylsilyl ort-butyldimethylsilyl ether; or an acetyl ester (—OC(═O)CH₃, —OAc). Forexample, an aldehyde or ketone group may be protected as an acetal orketal, respectively, in which the carbonyl group (>C═O) is converted toa diether (>C(OR)₂), by reaction with, for example, a primary alcohol.The aldehyde or ketone group is readily regenerated by hydrolysis usinga large excess of water in the presence of acid. For example, an aminegroup may be protected, for example, as an amide or a urethane, forexample, as: a methyl amide (—NHCO—CH₃); a benzyloxy amide(—NHCO—OCH₂C₆H₅, —NHCbz); as a t-butoxy amide (—NHCO—OC(CH₃)₃, —NH-Boc);a 2-biphenyl-2-propoxy amide (—NHCO—OC(CH₃)₂C₆H₄C₆H₅, —NH-Bpoc), as a9-fluorenylmethoxy amide (—NH-Fmoc), as a 6-nitroveratryloxy amide(—NH-Nvoc), as a 2-trimethylsilylethyloxy amide (—NH-Teoc), as a2,2,2-trichloroethyloxy amide (—NH-Troc), as an allyloxy amide(—NH-Alloc), as a 2(-phenylsulphonyl)ethyloxy amide (—NH-Psec); or, insuitable cases, as an N-oxide.

For example, a carboxylic acid group may be protected as an ester forexample, as: an C₁₋₇ alkyl ester (e.g., a methyl ester; a t-butylester); a C₁₋₇ haloalkyl ester (e.g., a C₁₋₇ trihaloalkylester); atriC₁₋₇ alkylsilyl-C₁₋₇ alkyl ester; or a C₅₋₂₀ aryl-C₁₋₇ alkyl ester(e.g., a benzyl ester; a nitrobenzyl ester); or as an amide, forexample, as a methyl amide.

For example, a thiol group may be protected as a thioether (—SR), forexample, as: a benzyl thioether; an acetamidomethyl ether(—S—CH₂NHC(═O)CH₃).

Synthesis of Coelenterazine Derivatives

Coelenterazine derivatives according to the present invention may besynthesized according those methods detailed in Examples 1-16.

Mutant Oplophorus Luciferases

In embodiments of the present invention, various techniques as describedherein were used to identify sites for amino acid substitution toproduce an improved synthetic OgLuc polypeptide. Additional techniqueswere used to optimize codons of the polynucleotides encoding the variouspolypeptides in order to enhance expression of the polypeptides. It wasfound that making one or more amino acid substitutions, either alone orin various combinations, produced synthetic OgLuc-type polypeptideshaving enhanced luminescence (e.g., enhanced brightness, enhanced signalstability, enhanced enzyme stability, and/or change in relativesubstrate specificity). Furthermore, including one or morecodon-optimizing substitutions in the polynucleotides which encode forthe various synthetic OgLuc variant polypeptides produced enhancedexpression of the polypeptides in various eukaryotic and prokaryoticexpression systems. One embodiment of the present invention is apolynucleotide that encodes a synthetic OgLuc variant polypeptide whichis soluble and active in the monomeric form when expressed inprokaryotic and/or eukaryotic cells.

The OgLuc variants of the present invention may be coupled to anyprotein of interest or molecule of interest. In some embodiments, thevariants are fusion proteins, for example some variants are coupled to aHaloTag® polypeptide attached at either the N-terminus or theC-terminus. Unless otherwise noted, the variants that are HaloTag®fusions include ‘HT7’ as part of the name, e.g., ‘IVY-HT7’. In someembodiments, a signal sequence (e.g., the naturally-occurring Oplophorusgracilirostris signal sequence) is attached to the N-terminus of thefusion protein to facilitate the secretion of the fusion protein fromthe cell. Signal sequences, other than the naturally-occurring signalsequence of OgLuc luciferase, are known in the art to facilitate proteinsecretion in mammalian cells or other cell types. Signal sequences, incombination with membrane anchoring sequences, may be used to positionor display OgLuc variants on the outer surface of the cellular membrane.Other methods, known in the art may also be used to position OgLucvariants to the membrane or other locations within the cell.

In some embodiments, the invention provides a modified decapodluciferase which has enhanced luminescence relative to a correspondingparental variant decapod luciferase. For example, the parental, variantOgLuc is C1+A4E, IVY, IV, QC27, QC27-9a, 9B8, 9B8 opt+K33N, 9B8opt+K33N+170G, V2 or “L27V”. In another embodiment, the inventionprovides a modified decapod luciferase which utilizes a novelcoelenterazine. In one embodiment, the modified decapod luciferase has achange in relative specificity for native, known or novelcoelenterazines. In one embodiment, the modified decapod luciferase hasa change in relative specificity relative to a corresponding parental,variant decapod luciferase.

In some embodiments, the corresponding parental, variant decapodluciferase is a decapod species, including various species from familieswithin the decapod order including, without limitation, luciferases ofthe Aristeidae family, including Plesiopenaeus coruscans; the Pandalideafamily, including Heterocarpus and Parapandalus richardi, theSolenoceridae family, including Hymenopenaeus debilis and Mesopenaeustropicalis; the Luciferidae family, including Lucifer typus; theSergestidae family, including Sergestes atlanticus, Sergestes arcticus,Sergestes armatus, Sergestes pediformis, Sergestes cornutus, Sergestesedwardsi, Sergestes henseni, Sergestes pectinatus, Sergestes sargassi,Sergestes similis, Sergestes vigilax, Sergia challengeri, Sergiagrandis, Sergia lucens, Sergia prehensilis, Sergia potens, Sergiarobusta, Sergia scintillans, and Sergia splendens; the Pasiphaeidaefamily, including Glyphus marsupialis, Leptochela bermudensis,Parapasiphae sulcatifrons, and Pasiphea tarda; the Oplophoridae family,including Acanthephyra acanthitelsonis, Acanthephyra acutifrons,Acanthephyra brevirostris, Acanthephyra cucullata, Acanthephyracurtirostris, Acanthephyra eximia, Acanthephyra gracilipes, Acanthephyrakingsleyi, Acanthephyra media, Acanthephyra microphthalma, Acanthephyrapelagica, Acanthephyra prionota, Acanthephyra purpurea, Acanthephyrasanguinea, Acanthephyra sibogae, Acanthephyra stylorostratis, Ephyrinabifida, Ephyrina figueirai, Ephyrina koskynii, Ephyrina ombango,Hymenodora glacialis, Hymenodora gracilis, Meningodora miccyla,Meningodora mollis, Meningodora vesca, Notostomus gibbosus, Notostomusauriculatus, Oplophorus gracilirostris, Oplophorus grimaldii, Oplophorusnovaezealandiae, Oplophorus spinicauda, Oplophorus foliaceus, Oplophorusspinosus, Oplophorus typus, Systellaspis braueri, Systellaspis cristata,Systellaspis debilis, and Systellaspis pellucida; and theThalassocaridae family, including Chlorotocoides spinicauda,Thalassocaris crinita, and Thalassocaris lucida. In certain embodiments,the modified luciferase has increased luminescence emission, e.g., atleast 1.3-fold, at least 2-fold, or at least 4-fold, in a prokaryoticcell and/or a eukaryotic cell relative to the corresponding wild-typeluciferase. In some embodiments, one or more properties of the modifieddecapod luciferase is compared to comparable properties of a luciferasefrom another species, e.g., a firefly luciferase or a Renillaluciferase.

In some embodiments, the OgLuc variant has at least 60%, e.g., at least65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%, or 100%, aminoacid sequence identity to SEQ ID NOs: 3, 5, 7, 9, 11, 13, 15, 17, 19,21, 27, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 56, 58, 60, 62, 64, 69,71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, or 95. In someembodiments, the OgLuc variant, or a functional fragment thereof, has nomore than 5 differences, or more preferably, no more than 4, 3, 2, or 1difference, or most preferably no differences, wherein the differencesoccur in positions corresponding to pattern position 1, 2, 3, 5, 8, 10,12, 14, 15, 17, or 18 of Formula (VII) according to Table 4. Differencesmay also include gaps or insertions between the pattern positions ofTable 4.

In some embodiments, the OgLuc variant of the invention has one or moreheterologous amino acid sequences at the N-terminus, C-terminus, or both(a fusion polypeptide such as one with an epitope or fusion tag), whichoptionally directly or indirectly interact with a molecule of interest.In some embodiments, the presence of the heterologous sequence(s) doesnot substantially alter the luminescence of the OgLuc variant eitherbefore or after the interaction with the molecule of interest. In someembodiments, the heterologous amino acid sequence is an epitope tag. Insome embodiments, the heterologous amino acid sequence is one which,during or after interaction with a molecule of interest, undergoes aconformational change, which in turn alters the activity of the OgLucvariant e.g., an OgLuc variant with such an amino acid sequence isuseful to detect allosteric interactions. The OgLuc variant or a fusionwith the OgLuc variant or a fragment thereof may be employed as areporter.

In some embodiments, a fragment of an OgLuc variant of the invention isfused to a heterologous amino acid sequence, the fusion thereby forminga beta-barrel, which fusion protein is capable of generatingluminescence from a naturally-occurring coelenterazine or an analogthereof including the various known coelenterazines discussed herein, ora novel coelenterazine of the present invention.

Also provided is a polynucleotide encoding an OgLuc variant of theinvention or a fusion thereof, an isolated host cell having thepolynucleotide or the OgLuc variant or a fusion thereof, and methods ofusing the polynucleotide, OgLuc variant or a fusion thereof or host cellof the invention.

The term “identity,” in the context of two or more nucleic acids orpolypeptide sequences, refers to two or more sequences or subsequencesthat are the same or have a specified percentage of amino acid residuesor nucleotides that are the same when compared and aligned for maximumcorrespondence over a comparison window or designated region as measuredusing any number of sequence comparison algorithms or by manualalignment and visual inspection. Methods of alignment of sequence forcomparison are well-known in the art. Optimal alignment of sequences forcomparison can be conducted by the algorithm of Smith et al., (J. Mol.Biol. 147:195-197 (1981)), by the homology alignment algorithm ofNeedleman and Wunsch, (J. Mol. Biol., 48:443-453 (1970)), by the searchfor similarity method of Pearson and Lipman, (Proc. Natl. Acad. Sci.USA, 85:2444-2448 (1988)), by computerized implementations of algorithmse.g., FASTA, SSEARCH, GGSEARCH (available at the University of VirginiaFASTA server by William R. Pearsonhttp://fasta.bioch.virginia.edu/fasta_www2/fasta_intro.shtml), theClustal series of programs (Chema et al., Nucl. Acids Res.31(13):3497-3500 (2003); available examples at http://www.ebi.ac.uk orhttp://www.ch.embnet.org), or other sequence analysis software. It isknown in the art that generating alignments with maximum correspondencebetween polypeptide sequences with significant sequence alterations(e.g., altered domain order, missing/added domains, repeated domains,shuffled domains, circular permutation) may involve the use ofspecialized methods, such as the ABA method (Raphael et al., Genome Res.14(11):2336-2346 (2004)), other suitable methods, or performing thealignment with two concatenated identical copies of the polypeptidesequences.

The term “nucleic acid molecule,” “polynucleotide” or “nucleic acidsequence” as used herein, refers to nucleic acid, including DNA or RNA,that comprises coding sequences necessary for the production of apolypeptide or protein precursor. The encoded polypeptide may be afull-length polypeptide, a fragment thereof (less than full-length), ora fusion of either the full-length polypeptide or fragment thereof withanother polypeptide, yielding a fusion polypeptide.

A polynucleotide encoding a protein or polypeptide means a nucleic acidsequence comprising the coding region of a gene, or in other words, thenucleic acid sequence encoding a gene product. The coding region may bepresent in a cDNA, genomic DNA or RNA form. When present in a DNA form,the oligonucleotide may be single stranded (e.g., the sense strand) ordouble stranded. Suitable control elements such as enhancers/promoters,splice junctions, polyadenylation signals, etc. may be placed in closeproximity to the coding region of the gene if needed to permit properinitiation of transcription and/or correct processing of the primary RNAtranscript. Other control or regulatory elements include, but are notlimited to, transcription factor binding sites, splicing signals,polyadenylation signals, termination signals and enhancer elements.

By “peptide,” “protein” and “polypeptide” is meant amino acid chains ofvarying lengths, regardless of post-translational modification (e.g.,glycosylation or phosphorylation). The nucleic acid molecules of theinvention encode a variant of a man made (i.e., synthetic) variantprotein or polypeptide fragment thereof, which has an amino acidsequence that is at least 60%, e.g., at least 65%, 70%, 75%, 80%, 85%,90%, 95%, 96%, 97%, 98%, 99%, or 100%, amino acid sequence identity tothe amino acid sequence of the parental protein from which it isderived, where the parental protein can be a naturally-occurring (nativeor wild-type) sequence or a variant sequence which is subsequentlymodified further. The term “fusion polypeptide” or “fusion protein”refers to a chimeric protein containing a reference protein (e.g., OgLucvariant) joined at the N- and/or C-terminus to one or more heterologoussequences (e.g., a non-OgLuc polypeptide). The heterologous sequence caninclude, but is not limited to, reporter proteins such as the HALOTAG®fusion protein (Promega Corp.), FlAsH (fluorescein arsenical helixbinder), and ReAsH (red arsenical helix binder) (e.g., LUMIO tagrecognition sequence (Invitrogen)), chloramphenicol acetyltransferase(CAT), β-galactosidase (β-Gal), lactamase (P-gal), neomycin resistance(Neo), GUS, galactopyranoside, green fluorescent protein (GFP),luciferase (e.g., a Renilla reniformis luciferase, a firefly luciferase(e.g., Photinus pyralis or Photuris pennsylvanica), or a click beetleluciferase (e.g., Pyrophorus plagiophthalamus or Pyrearinustermitilluminans) or a glowworm luciferase (e.g., Phrixothrix hirtus),xylosidase, thymidine kinase, arabinosidase and SNAP-tag, CLIP-tag,ACP-tag and MCP-tag (New England Biolabs). In one embodiment, a chimericprotein contains an OgLuc variant joined at the N-terminus to a HALOTAG®fusion protein (Promega Corp.). In another embodiment, a chimericprotein contains an OgLuc variant joined at the C-terminus to a HALOTAG®fusion protein.

Nucleic acids are known to contain different types of “mutations”, whichrefers to an alteration in the sequence of a nucleotide at a particularbase position relative to the wild-type sequence. Mutations may alsorefer to insertion or deletion of one or more bases so that the nucleicacid sequence differs from a reference, e.g., a wild-type sequence, orreplacement with a stop codon. A “substitution” refers to a change in anamino acid at a particular position in a sequence, e.g., a change from Ato E at position 4.

The term “vector” refers to nucleic acid molecules into which fragmentsof DNA may be inserted or cloned and can be used to transfer DNAsegment(s) into a cell and capable of replication in a cell. Vectors maybe derived from plasmids, bacteriophages, viruses, cosmids, and thelike.

The term “wild-type” or “native” as used herein, refers to a gene orgene product that has the characteristics of that gene or gene productisolated from a naturally occurring source. A wild-type gene is thatwhich is most frequently observed in a population and is thusarbitrarily designated the “wild-type” form of the gene. In contrast,the term “mutant” refers to a gene or gene product that displaysmodifications in sequence and/or functional properties (i.e., alteredcharacteristics) when compared to the wild-type gene or gene product. Itis noted that naturally occurring mutants can be isolated; these areidentified by the fact that they have altered characteristics whencompared to the wild-type gene or gene product.

Exemplary Polynucleotides and Proteins

The invention includes an OgLuc variant or protein fragments thereof,e.g., those with deletions, for instance a deletion of 1 to about 5residues, and chimeras (fusions) thereof (see U.S. Patent PublicationNo. 2009/0253131 and WIPO Publication No. WO 2007/120522, thedisclosures of which are incorporated by reference herein) having atleast one amino acid substitution relative to a wild-type OgLuc, whichsubstitution results in the OgLuc variant having enhanced stability,enhanced luminescence, e.g., increased luminescence emission, greaterstability of the luminescence kinetics, and/or altered luminescencecolor. The sequences of an OgLuc variant are substantially the same asthe amino acid sequence of a corresponding wild-type OgLuc. Apolypeptide or peptide having substantially the same sequence means thatan amino acid sequence is largely, but is not entirely, the same andretains the functional activity of the sequence to which it is related.In general, two amino acid sequences are substantially the same if theyare at least 60%, e.g., at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%,97%, 98%, or 99%, but less than 100%, amino acid sequence identity. Insome embodiments, the OgLuc variant is encoded by a recombinantpolynucleotide. In some embodiments, the OgLuc variant, or a functionalfragment thereof, has no more than 5 differences, or more preferably nomore than 4, 3, 2, or 1 difference, or most preferably no differences,wherein the differences occur in positions corresponding to patternposition 1, 2, 3, 5, 8, 10, 12, 14, 15, 17, or 18 of Formula (VII)according to Table 4. Differences may also include gaps, insertions, orpermutations between the pattern positions of Table 4.

The OgLuc variant proteins or fusion proteins of the invention may beprepared by recombinant methods or by solid phase chemical peptidesynthesis methods. Such methods are known in the art.

Methods of Use and Kits

The compounds and proteins of the invention may be used in any way thatluciferases and luciferase substrates, e.g., coelenterazines, have beenused. For example, they may be used in a bioluminogenic method whichemploys an analog of coelenterazine to detect one or more molecules in asample, e.g., an enzyme, a cofactor for an enzymatic reaction, an enzymesubstrate, an enzyme inhibitor, an enzyme activator, or OH radicals, orone or more conditions, e.g., redox conditions. The sample may includean animal (e.g., a vertebrate), a plant, a fungus, physiological fluid(e.g., blood, plasma, urine, mucous secretions and the like), a cell, acell lysate, a cell supernatant, or a purified fraction of a cell (e.g.,a subcellular fraction). The presence, amount, spectral distribution,emission kinetics, or specific activity of such a molecule may bedetected or quantified. The molecule may be detected or quantified insolution, including multiphasic solutions (e.g., emulsions orsuspensions), or on solid supports (e.g., particles, capillaries, orassay vessels). In some embodiments the OgLuc variant can be used inluminescence-based assays to detect an enzyme of interest, e.g., CYP450enzyme, MAO A or B enzyme, a caspase, etc. The novel coelenterazinescould be used with photoproteins such as aequorin, obelin, or iPhotina.In some embodiment, the OgLuc variant can be used as an energy donor toanother molecule (e.g., to a fluorophore, a chromophore, or ananoparticle).

The invention also provides a polynucleotide encoding a transcriptionalreporter. In some embodiments, the OgLuc variant or fragment thereofcould be operably linked to transcription regulatory sequences, e.g.,one or more enhancer, a promoter, a transcription termination sequenceor a combination thereof, to form an expression cassette. For example,the OgLuc variant could be operably linked to a minimal promoter and acAMP-response element (CRE).

The proteins of the invention may be used as biosensors, e.g., an OgLucvariant, which, in the presence of another molecule (e.g, one or moremolecules of interest), or under certain conditions, has one or morealtered activities. Upon interacting with a molecule of interest orbeing subject to certain conditions, the biosensor undergoes aconformational change or is chemically altered which causes analteration of the enzyme activity or luminescence, e.g., specificactivity, spectral distribution, or emission kinetics. For example, theOgLuc variant of the present invention, for example a circularlypermuted variant, can comprise an interaction domain for a molecule ofinterest. Alternatively, for example, the OgLuc variant may be coupledto an energy acceptor, for example a fluorescent protein, and comprisean interaction domain that alters the efficiency of energy transfer fromthe enzyme to the energy acceptor. For example, the biosensor could begenerated to detect proteases, kinases, a ligand, a binding protein suchas an antibody, cyclic nucleotides such as cAMP or cGMP, or a metal suchas calcium, by insertion of a suitable sensor region into the OgLucvariant sequence. One or more sensor region can be inserted at theC-terminus, the N-terminus, and/or at one or more suitable location inthe polypeptide sequence, where the sensor region comprises one or moreamino acids. In the case of a circularly-permuted OgLuc variant, thesensor region may be inserted between the N- and C-termini of the parentOgLuc variant. In addition, one or all of the inserted sensor regionsmay include linker amino acids to couple the sensor to the remainder ofthe OgLuc variant polypeptide. Examples of luciferase biosensors aredisclosed in U.S. Pat. Appl. Publ. Nos. 2005/0153310 and 2009/0305280and PCT Publ. No. WO 2007/120522 A2, each of which is incorporated byreference herein.

In various embodiments, the OgLuc variants disclosed herein may be usedto transfer energy to an energy acceptor, for example in BioluminescenceResonance Energy Transfer (BRET) analysis. For example, the OgLucvariants used in BRET analysis can be used to determine if two moleculesare capable of binding each other or co-localize in a cell. For example,an OgLuc variant can be used as a bioluminescence donor molecule whichis combined with a molecule or protein of interest to create a firstfusion protein. In various embodiments, the first fusion proteincontains an OgLuc variant and a protein of interest. In variousembodiments, the first fusion proteins containing the OgLuc variant canbe used in BRET analysis to detect protein/protein interaction insystems including but not limited to cell lysates, intact cells, andliving animals. In various embodiments, HALOTAG® can be used as afluorescent acceptor molecule. In some embodiments, HALOTAG® can befused to a second protein of interest or to an OgLuc variant. Forexample, an OgLuc variant can be fused to HALOTAG®, expressed in cellsor animals, and labeled with a fluorescent HALOTAG® ligand such asHALOTAG® TMR ligand. The fusion can subsequently be excited to fluorescein the presence of a cell-permeant OgLuc substrate. In some embodiments,BRET may be performed using OgLuc variants in combination withfluorescent proteins, including but not limited to Green FluorescentProtein (GFP) or Red Fluorescent Protein (RFP) or fluorescent labelsincluding fluorescein, rhodamine green, Oregon green, or Alexa 488, toname a few non-limiting examples.

In various embodiments, the OgLuc variants and/or the novelcoelenterazines of the present invention may be used in proteincomplementation assays (PCA) to detect the interaction of twobiomolecules, e.g., polypeptides. For example, an OgLuc variant of thepresent invention can be separated into two fragments at a site(s)tolerant to separation and each fragment of the separated OgLuc variantcan be fused to one of a pair of polypeptides of interest believed tointeract, e.g., FKBP and FRB. If the two polypeptides of interest do infact interact, the OgLuc fragments then come into close proximity witheach other to reconstitute the functional, active OgLuc variant. In someembodiments, the activity of the reconstituted OgLuc variant can then bedetected and measured using a native or known coelenterazine or a novelcoelenterazine of the present invention. In some embodiments, the splitOgLuc variant can be used in a more general complementation systemsimilar to lac-Z (Langley et al., PNAS 72:1254-1257 (1975)) orribonuclease S (Levit and Berger, J. Biol. Chem. 251:1333-1339 (1976)).In some embodiments, an OgLuc variant fragment (designated “A”) known tocomplement with another OgLuc variant fragment (“B”) can be fused to atarget protein, and the resulting fusion can be monitored vialuminescence in a cell or cell lysate containing fragment B. In someembodiments, the source of fragment B could be the same cell (e.g., ifthe gene for fragment B is integrated into the genome of the cell or iscontained on another plasmid within the cell) or it could be a lysate orpurified protein derived from another cell. In some embodiments, thissame fusion protein (fragment A) could be captured or immobilized usinga fusion between fragment B and a polypeptide such as HALOTAG® capableof attachment to a solid support. In some embodiments, luminescence canbe used to demonstrate successful capture or to quantify the amount ofmaterial captured.

In various embodiments, the OgLuc variants and/or the novelcoelenterazines of the present invention may be used to quantifycoelenterazine. In some embodiments, a coelenterazine (e.g., a native orknown coelenterazine, or a novel coelenterazine of the presentinvention) can be used as a probe of a specific biochemical activity,e.g., apoptosis and drug metabolism. In some embodiments, thecoelenterazine concentration is coupled to a specific enzyme activity bya “pro-coelenterazine” or “pro-substrate” that can be acted on by thespecific enzyme of interest. In some embodiments, the pro-coelenterazineis a molecule that cannot support luminescence directly when combinedwith luciferase, but can be converted into coelenterazine throughcatalytic processing by a specific enzyme of interest. In someembodiments, the approach can be used for enzymes such as those used indrug metabolism, e.g., cytochrome P450 enzymes, monoamine oxidase, andglutathione S-transferase; and apoptosis, e.g., caspases. For example,coelenterazine (e.g., a native or known coelenterazine, or a novelcoelenterazine of the present invention) can be modified to contain acleavable group, such as 6′-O-methyl. In some embodiments, whenincubated with a specific cytochrome P450 enzyme, the 6′O-methyl iscleaved, and the pro-coelenterazine converted to coelenterazine whichcan be detected with an OgLuc variant of the present invention. In someembodiments, the pro-coelenterazine can be combined with othercomponents necessary to support luminescence, e.g., luminescent proteinsuch as an OgLuc variant of the present invention, to provide a singlereagent and a homogeneous assay. For example, when the reagent is addedto a sample, luminescence is generated as pro-coelenterazine isconverted to coelenterazine. In various embodiments, similar assays canbe developed for other enzymes, small molecules, or other cellularprocesses that can be linked to the generation of coelenterazines frompro-coelenterazines.

In various embodiments, the OgLuc variants and/or the novelcoelenterazines of the present invention may be used as genetictranscriptional reporter systems. In some embodiments, the OgLucvariants can be multiplexed with a luciferase that emits light at adifferent wavelength, e.g., red click beetle luciferase (CHROMA-LUC™;Promega Corp.). For example, if an OgLuc variant of the presentinvention is used as a functional reporter, then the red CHROMA-LUC™luciferase could be used to control for non-specific effects on geneticregulation or to normalize for transfection efficiency. In someembodiments, luminescence generated from the OgLuc variant(approximately 460 nm) and red CHROMA-LUC™ (approximately 610 nm) can beeasily resolved using a luminometer with wavelength-discriminatingfilters, enabling the measurement of both signals from the same sample.In another example, an OgLuc variant of the present invention could beused as a transcriptional reporter and paired with a luciferase thatemits light at a different wavelength contained in an assay reagent. Forexample, an OgLuc variant of the present invention could be used astranscriptional reporter and paired with either aequorin or a cAMPcircularly-permuted firefly luciferase biosensor, or bothsimultaneously, to detect multiple pathways in a single sample. In sucha system, for example, aequorin could be used for the detection and/ormeasurement of calcium, the biosensor for the detection and/ormeasurement of cAMP, and an OgLuc variant for monitoring of downstreamgene expression. In another example, an OgLuc variant may be used withone or more additional luciferases, where the luminescence of eachluciferase may be separately measured through the use of selectiveenzyme inhibitors. For example, the luminescence of a first luciferasemay be measured upon addition of appropriate substrates and buffers,followed by measurement of a second luciferase upon a subsequentaddition of appropriate substrates and buffers and one or moreinhibitors selective for the first luciferase. In another example, theluciferase contained in an assay reagent may be used for measuring aspecific aspect of cellular physiology, for example ATP to estimate cellviability, or caspase activity to estimate cellular apoptosis.

In various embodiments, the OgLuc variants of the present invention maybe used as reporters in difficult to transfect cell lines or perhapseven in non-dividing primary cells, e.g., stem cells or HepG2 cells. Dueto their high signal intensity, the OgLuc variants of the presentinvention will enable detectable luminescence when transfectionefficiency is low. In some embodiments, the OgLuc variants can be usedas reporters in cells that are especially sensitive to conditionsassociated with transfection, e.g., which are sensitive to elevated DNAconcentrations or the addition of transfection reagent. Thus, in variousembodiments, due to the enhanced luminescence of the OgLuc variants ofthe present invention, an adequate level of luminescence can be achievedusing lower DNA concentrations, less transfection reagent, and/orshorter post-transfection times prior to beginning an assay so thatthere is a reduced toxicity burden on sensitive cells. In variousembodiments, the enhanced luminescence of the OgLuc variants will alsoallow for a signal to be detected at much later time points. In stillother embodiments, the OgLuc variants could be used as reporters forsingle-copy native promoters.

In various embodiments, the OgLuc variants of the present invention maybe used as fusion tags for a target protein of interest as a way tomonitor intracellular levels of the target protein. In some embodiments,the OgLuc variants can be used to monitor specific proteins involved instress response pathways (e.g., DNA damage, oxidative stress,inflammation) in cells as a way to probe the role various types ofstimuli may play in these pathways. In some embodiments, the OgLucvariants can also be used as a means to monitor cellular trafficking ofa target protein. For example, the OgLuc variants can also be fused toviral genomes (e.g., HIV, HCV) so that titer levels, i.e., infectivity,can be monitored in cells following treatment with potential antiviralagents. In some embodiments, the variants can also be fused to greenfluorescent protein (GFP) or HALOTAG® (in addition to a target protein)for fluorescence activated cell sorting (FACS) to identify highexpression clones.

In various embodiments, identification of robust, stable cell linesexpressing an OgLuc variant of the present invention, either in thecytoplasm or as a secreted form, can be facilitated by the enhancedsignal of the OgLuc variant and the small size of the OgLuc gene. Therelatively small gene sequence should reduce the likelihood of geneticinstability resulting from the integration of the foreign DNA into acell's genome.

In various embodiments, the OgLuc variants of the present invention canbe integrated into a variety of different immunoassay concepts. Forexample, an OgLuc variant can be fused to a primary or secondaryantibody to provide a method of detection for a particular analyte. Asanother example, an OgLuc variant can be fused to protein A or proteinG, and the fusion could then be used to detect a specific antibody boundto a particular analyte. As another example, an OgLuc variant can befused to streptavidin and used to detect a specific biotinylatedantibody bound to a particular analyte. As yet another example,complementary fragments of an OgLuc variant can be fused to primary andsecondary antibodies, where the primary antibody recognizes a particularanalyte, and the secondary antibody recognizes the primary antibody. Insome embodiments, the OgLuc variant activity would be reconstituted inthe presence of analyte. As still another example, an OgLuc variant canbe conjugated to an analyte (e.g., prostaglandins) and used in acompetitive sandwich ELISA format. The OgLuc variant conjugated to ananalyte may also be used to detect antibodies capable of binding theanalyte, where the binding activity allows the OgLuc variant to beselectively linked to the antibody. An example using Renilla luciferasefor quantitatively measuring patient antibody titers to an antigenictarget is the Luciferase Immunoprecipitation System (Burbelo et al.,Expert Review of Vaccines 9(6):567-578 (2010))

In various embodiments, the OgLuc variants and novel substrates of thepresent invention can be used for detecting luminescence in live cells.In some embodiments, an OgLuc variant can be expressed in cells (as areporter or otherwise), and the cells treated with a coelenterazine,e.g., a novel coelenterazine such as PBI-3939, which will permeate cellsin culture, react with the OgLuc variant and generate luminescence. Inaddition to being cell permeant, PBI-3939 shows comparablebiocompatibility to native coelenterazine in terms of cell viability. Insome embodiments, a version of PBI-3939 containing chemicalmodifications known to increase the stability of native coelenterazinein media can be synthesized and used for more robust, live cell OgLucvariant-based reporter assays. In still other embodiments, a sample(including cells, tissues, animals, etc.) containing an OgLuc variantand/or a novel coelenterazine of the present invention may be assayedusing various microscopy and imaging techniques. In still otherembodiments, a secretable OgLuc variant is expressed in cells as part ofa live-cell reporter system.

In various embodiments, the OgLuc variants and/or novel coelenterazinesdisclosed herein may be provided as part of a kit. The kit may includeone or more OgLuc variants as disclosed herein (in the form of apolypeptide, a polynucleotide, or both) and/or a coelenterazine, alongwith suitable reagents and instructions to enable a user to performassays such as those disclosed herein. The coelenterazine may be any ofthe native, known, or novel coelenterazines disclosed herein. The kitmay also include one or more buffers, such as those disclosed herein.

Vectors and Host Cells Encoding the Modified Luciferase or FusionsThereof

Once a desirable nucleic acid molecule encoding an OgLuc variant or afragment thereof, such as one with luminescence activity or which may becomplemented by another molecule to result in luminescence activity, ora fusion thereof with luminescence activity, is prepared, an expressioncassette encoding the OgLuc variant or a fragment thereof, e.g., one forcomplementation, or a fusion thereof with luminescence activity, may beprepared. For example, a nucleic acid molecule comprising a nucleic acidsequence encoding an OgLuc variant is optionally operably linked totranscription regulatory sequences, e.g., one or more enhancers, apromoter, a transcription termination sequence or a combination thereof,to form an expression cassette. The nucleic acid molecule or expressioncassette may be introduced to a vector, e.g., a plasmid or viral vector,which optionally includes a selectable marker gene, and the vectorintroduced to a cell of interest, for example, a prokaryotic cell suchas E. coli, Streptomyces spp., Bacillus spp., Staphylococcus spp. andthe like, as well as eukaryotic cells including a plant (dicot ormonocot), fungus (including yeast, e.g., Pichia, Saccharomyces orSchizosaccharomyces), or a mammalian cell, lysates thereof, or to an invitro transcription/translation mixture. Mammalian cells include but arenot limited to bovine, caprine, ovine, canine, feline, non-humanprimate, e.g., simian, and human cells. Mammalian cell lines include,but are not limited to, CHO, COS, HEK293, HeLa, CV-1, SH-SY5Y, and NM3T3 cells, although numerous other cell lines can also be used as well.

The expression of an encoded OgLuc variant may be controlled by anypromoter capable of expression in prokaryotic cells or eukaryotic cellsincluding synthetic promoters. Prokaryotic promoters include, but arenot limited to, SP6, T7, T5, tac, bla, trp, gal, lac or maltosepromoters, including any fragment that has promoter activity. Eukaryoticpromoters include, but are not limited to, constitutive promoters, e.g.,viral promoters such as CMV, SV40 and RSV promoters, as well asregulatable promoters, e.g., an inducible or repressible promoter suchas the tet promoter, the hsp70 promoter and a synthetic promoterregulated by CRE, including any fragment that has promoter activity. Theexpression of an encoded OgLuc variant may also be controlled bypost-transcriptional processes, such as by regulation of RNA processingor regulation of translation, for example by RNAi, miRNA, shRNA, siRNA,or by RNA or protein degradation. The nucleic acid molecule, expressioncassette and/or vector of the invention may be introduced to a cell byany method including, but not limited to, calcium-mediatedtransformation, electroporation, microinjection, lipofection, and thelike.

Optimized Sequences, and Vectors and Host Cells Encoding the OgLucVariants

Also provided is an isolated nucleic acid molecule (polynucleotide)comprising a nucleic acid sequence encoding an OgLuc variant of theinvention, a functional fragment thereof or a fusion protein thereof. Insome embodiments, the isolated nucleic acid molecule comprises a nucleicacid sequence which is optimized for expression in at least one selectedhost. Optimized sequences include sequences which are codon optimized,i.e., codons which are employed more frequently in one organism relativeto another organism, e.g., a distantly related organism, as well asmodifications to add or modify Kozak sequences and/or introns, and/or toremove undesirable sequences, for instance, potential transcriptionfactor binding sites. Such optimized sequences can provide enhancedexpression, e.g., increased levels of protein expression, whenintroduced into a host cell. Examples of optimized sequences aredisclosed in U.S. Pat. No. 7,728,118 and U.S. Pat. Appl. Publ. Nos.2008/0070299, 2008/0090291, and 2006/0068395, each of which isincorporated by reference herein.

In some embodiments, the polynucleotide includes a nucleic acid sequenceencoding an OgLuc variant of the invention, which nucleic acid sequenceis optimized for expression in a mammalian host cell. In someembodiments, an optimized polynucleotide no longer hybridizes to thecorresponding non-optimized sequence, e.g., does not hybridize to thenon-optimized sequence under medium or high stringency conditions. Theterm “stringency” is used in reference to the conditions of temperature,ionic strength, and the presence of other compounds, under which nucleicacid hybridizations are conducted. With “high stringency” conditions,nucleic acid base pairing will occur only between nucleic acid fragmentsthat have a high frequency of complementary base sequences. Thus,conditions of “medium” or “low” stringency are often used when it isdesired that nucleic acids that are not completely complementary to oneanother be hybridized or annealed together. The art knows well thatnumerous equivalent conditions can be employed to comprise medium or lowstringency conditions.

In some embodiments, the polynucleotide has less than 90%, e.g., lessthan 80%, nucleic acid sequence identity to the correspondingnon-optimized sequence and optionally encodes a polypeptide having atleast 60%, e.g., at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%,98%, 99%, or 100%, amino acid sequence identity with the polypeptideencoded by the non-optimized sequence. Constructs, e.g., expressioncassettes, and vectors comprising the isolated nucleic acid molecule,e.g., with optimized nucleic acid sequence, as well as kits comprisingthe isolated nucleic acid molecule, construct or vector are alsoprovided.

A nucleic acid molecule comprising a nucleic acid sequence encoding anOgLuc variant of the invention, a fragment thereof or a fusion thereofis optionally optimized for expression in a particular host cell andalso optionally operably linked to transcription regulatory sequences,e.g., one or more enhancers, a promoter, a transcription terminationsequence or a combination thereof, to form an expression cassette.

In some embodiments, a nucleic acid sequence encoding an OgLuc variantof the invention, a fragment thereof or a fusion thereof is optimized byreplacing codons, e.g., at least 25% of the codons in a parental OgLucsequence with codons which are preferentially employed in a particular(selected) cell. Preferred codons have a relatively high codon usagefrequency in a selected cell, and preferably their introduction resultsin the introduction of relatively few transcription factor binding sitesfor transcription factors present in the selected host cell, andrelatively few other undesirable structural attributes. Examples ofundesirable structural attributes include, but not limited to,restriction enzyme sites, eukaryotic sequence elements, vertebratepromoter modules and transcription factor binding sites, responseelements, E. coli sequence elements, mRNA secondary structure. Thus, theoptimized nucleic acid product may have an improved level of expressiondue to improved codon usage frequency, and a reduced risk ofinappropriate transcriptional behavior due to a reduced number ofundesirable transcription regulatory sequences.

An isolated and optimized nucleic acid molecule may have a codoncomposition that differs from that of the corresponding wild-typenucleic acid sequence at more than 30%, 35%, 40% or more than 45%, e.g.,50%, 55%, 60% or more of the codons. Exemplary codons for use in theinvention are those which are employed more frequently than at least oneother codon for the same amino acid in a particular organism and, insome embodiments, are also not low-usage codons in that organism and arenot low-usage codons in the organism used to clone or screen for theexpression of the nucleic acid molecule. Moreover, codons for certainamino acids (i.e., those amino acids that have three or more codons),may include two or more codons that are employed more frequently thanthe other (non-preferred) codon(s). The presence of codons in thenucleic acid molecule that are employed more frequently in one organismthan in another organism results in a nucleic acid molecule which, whenintroduced into the cells of the organism that employs those codons morefrequently, is expressed in those cells at a level that is greater thanthe expression of the wild-type or parent nucleic acid sequence in thosecells.

In some embodiments of the invention, the codons that are different arethose employed more frequently in a mammal, while in still otherembodiments, the codons that are different are those employed morefrequently in a plant. Preferred codons for different organisms areknown to the art, e.g., see http://www.kazusa.or.jp./codon/. Aparticular type of mammal, e.g., a human, may have a different set ofpreferred codons than another type of mammal. Likewise, a particulartype of plant may have a different set of preferred codons than anothertype of plant. In one embodiment of the invention, the majority of thecodons that differ are ones that are preferred codons in a desired hostcell. Preferred codons for organisms including mammals (e.g., humans)and plants are known to the art (e.g., Wada et al., Nucl. Acids Res.,18:2367 (1990); Murray et al., Nucl. Acids Res., 17:477 (1989)).

EXAMPLES Reference Example 1 Synthesis of α-Aminonitrile (Compound 1)

A flask was charged with sodium bisulfite (71.4 mmol) and 17 mL ofwater. To this, a solution of aldehyde (69.3 mmol) in 14 mL oftetrahydrofuran (THF) was added dropwise at a rate that kept theinternal temperature below 60° C. The resulting suspension was stirredat ambient temperature for 40 min, and ammonium hydroxide solution (4.85mL) added over 2 min. The resulting solution was magnetically stirredwhile being heated in an oil bath at 60° C. for 1 hr and then left atambient temperature overnight. The solution was cooled in anice/saltwater bath until the internal temperature measured below 5° C.To this, a solution of sodium cyanide (71.4 mmol) in 14 mL of water wasadded dropwise over 30 min. The resulting mixture was stirred atapproximately 10° C. for 20 min, 30° C. for 2 hrs, and at ambienttemperature for 18 hrs. The reaction mixture was extracted into three200 mL portions of diethyl ether, and the combined extracts dried overanhydrous sodium sulfate. The mixture was filtered, and the solutioncooled in an ice bath for 20 min. To the stirred solution, hydrogenchloride gas was added until precipitation ceased, and the suspensionstirred for 1 hr. The solid was isolated by filtration and rinsed withthree 50 mL portions of diethyl ether. The material was dried undervacuum, and 6.4 g (47.5 mmol) of a white solid was obtained (69%).Procedure was adapted from: Freifelder and Hasbrouck, “Synthesis ofPrimary 1,2-Diamines by Hydrogenation of alpha-Aminonitriles,” Journalof the American Chemical Society, 82(3):696-698 (1960).

Reference Example 2 Synthesis of 2-oxo-2-phenylacetaldehyde oxime(Compound 2)

A flask was charged with potassium tert-butoxide (58 mmol) and 63 mL oftert-butyl alcohol. The mixture was stirred until a solution was formed,and a solution of the appropriate benzophenone (50 mmol) in 35 mL oftert-butyl alcohol added dropwise over 15 min. The reaction mixture wasstirred for 1 hr, and the neat isoamyl nitrite (75 mmol) added over fivemin. The reaction mixture was monitored for completion and then dilutedwith 100 mL of heptanes. The resulting solid (38 mmol) was collected viasuction filtration and dried to a constant weight under vacuum.Procedure was adapted from: Hagedorn et al., Chem. Ber., 98:193 (1965).

Reference Example 3 Synthesis of Pyrazine Derivatives (Compound 3)

A 3-neck flask was fitted with a thermometer, septum, and argon line. Tothis, aminonitrile (47.5 mmol), dry pyridine (190 mL), and oxime (61.75mmol) was added. The mixture was well stirred for 15 min, andtetra-chloro(bis-pyridyl)titanium complex (94.9 mmol) added in fiveportions over 35 min making sure the internal temperature remained below40° C. After the addition was complete, the reaction mixture was stirredovernight at ambient temperature. The reaction mixture was slowly addedto a solution of sodium bicarbonate (21.75 g in 174 mL water) in smallportions. The resulting mixture was well stirred for 15 min and 80 g ofcelite was added. The suspension was stirred for 30 min and filteredthrough a Buchner funnel. The filtrate was removed to a separatoryfunnel, and the filter cake was suspended in 400 mL of methanol. Themixture was stirred for 30 min and filtered again. This process wasrepeated a total of four times. The methanolic filtrates were combinedand concentrated, and the residue dissolved in 200 mL of ethyl acetate(EtOAc). The solution was added to the separatory funnel containing theoriginal filtrate, and the mixture further extracted with three 100 mLportions of EtOAc. The combined extracts were washed with two 100 mLportions of saturated sodium carbonate and two 100 mL portions of brinesolution. The organic solvent was evaporated, and the crudepyazine-oxide obtained as a brown oil. The material was dissolved in 3mL of methanol, and 89 mL of dichloromethane (DCM) was added. To thissolution, zinc dust (80.7 mmol) was added, and the mixture cooled in anice bath until an internal temperature of 15° C. was reached. Themixture was treated with glacial acetic acid (3 mL) and warmed to aninternal temperature of 30° C. in an oil bath for 40 min. The reactionmixture was cooled to room temperature and filtered through a pad ofcelite. The filter cake was rinsed with DCM, and the combined filtrateswashed with an aqueous solution of saturated sodium bicarbonate. Thecrude product was purified by chromatography over silica gel using aheptane/EtOAc gradient. This gave 2.9 g (29%) of the pyrazine as a brownsolid. Procedure was adapted from: Kishi et al., “The structureconfirmation of the light-emitting moiety of bioluminescent jellyfish.”Tetrahedron Lett., 13(27):2747 (1972).

Reference Example 4 Synthesis of Coelenterazines

Method A: (the following compounds can be synthesized by Method A:compounds PBI-3840, PBI-3886, PBI-3857, PBI-3887, PBI-3913, PBI-3894,PBI-3896, PBI-3897, PBI-3841 and PBI-3842)

A flask was charged with pyrazine (8.25 mmol), pyruvic acid (14.0 mmol),camphor sulfonic acid (0.8 mmol), and anhydrous 2-methyl THF (150 mL).The flask was equipped with a condenser and soxhlet extractor chargedwith 4-angstrom molecular sieves, and the reaction mixture heated in anoil bath at 110° C. for 18 hrs. The sieves were replaced with freshones, and reflux continued for 24 hrs. The reaction mixture was filteredand concentrated, and the residue dissolved in EtOAc (200 mL). Thissolution was washed with three 25 mL portions of saturated sodiumbicarbonate solution, 100 mL of 0.1 M sodium acetate buffer, pH 5, and100 mL of brine solution. The solution was dried over magnesium sulfate,filtered, and concentrated to give 2.3 g (6.2 mmol, 75%) of the crudeenamine/acid. This material was dissolved in anhydrous THF (30 mL), andthe solution cooled in an ice/water bath for 10 min. To this, thecarbodiimide (9.0 mmol) and neat diisopropylethyl amine (14.9 mmol) wasadded. The cold bath was removed after 10 min, and the reaction mixturestirred at ambient temperature for 3 hrs. To the reaction mixture, 50 mLof 0.1 M sodium acetate buffer, pH 5 was added, and the mixture wellstirred for 10 min. The biphasic mixture was extracted with three 100 mLportions of EtOAc, and the combined extracts washed with brine solution.The organic solution was concentrated, and the residue purified bychromatography over silica gel using a DCM/methanol gradient. This gave336 mg (0.94 mmol, 16%) of the dehydrocoelenterazine as a red solid.This material was suspended in 10 mL of methanol, and the mixture cooledin an ice bath. To this, sodium borohydride (100 mg, 2.6 mmol) was addedin three portions over 1 hr. The reaction mixture was stirred for anadditional 30 min, and neat glacial acetic acid added drop wise until apH of 5 was reached. The solution was concentrated, and the residuetriturated with 15 mL of water. The solid was isolated via suctionfiltration and dried under vacuum for several hours to give 318 mg (94%)of the crude coelenterazine as a yellow solid. Procedure was adaptedfrom: Kakoi and Inoue, Chem. Lett. 11(3):299-300 (1980).

Method B: (the following compounds can be synthesized by method B:compounds PBI-3882, PBI-3932, PBI-3881)

A flask was charged with the glyoxal (2.2 mmol), aminopyrazine (1.1mmol), ethanol (20 mL), 12N HCl (0.6 mL), and water (1 mL). The reactionmixture was heated at reflux for 24 hrs and concentrated. The residuewas purified by column chromatography over silica gel using aDCM/methanol gradient. This gave 100 mg (0.25 mmol, 23%) of thecoelenterazine product as a dark solid. Procedure was adapted from:Inoue et al. “Squid bioluminescence. II. Isolation from Wataseniascintillans and synthesis of2-(p-hydroxybenzyl)-6-(p-hydroxyphenyl)-3,7-dihydroimidazo[1,2-a]pyrazin-3-one.”Chem. Lett., 4(2): 141-4 (1975).

Method C: Synthesis of novel coelenterazines (the following compoundscan be synthesized by method C: PBI-3939, PBI-3945, PBI-3889, PBI-4002)

The compound 4-(5-amino-6-benzylpyrazin-2-yl)phenol can be preparedaccording to previously described methods (Kishi et al., TetrahedronLett., 13:2747 (1972); Mosrin et al., Organic Letters, 11:3406 (2009);Kakoi, Chem. Pharm. Bull., 50:301 (2002)).

Synthesis of 2-amino-3-benzyl-5-phenylpyrazine. A round bottomed flaskwas charged with 5 g (33.5 mmol) of 2-isonitrosoacetophenone, 6.7 g(36.8 mmol) of 2-amino-3-phenylpropanenitrile hydrochloride and 100 mLof dry pyridine. The mixture was cooled to −20° C. and 4.6 mL (40.0mmol) of TiCl₄ was added dropwise. The reaction was kept at −20° C. for30 min and heated to 80° C. for 2.5 hrs. The solvent was evaporated, andthe residue taken up in 1 L of DCM. This solution was washed withsaturated NaHCO₃ and brine. All volatiles were evaporated, and theresidue redissolved in ethanol (400 mL). Raney Ni (2.0 g, aqueoussuspension) was added, and the reaction allowed to stir for 5 days under1 atm of hydrogen. The mixture was passed through celite, and volatilesremoved. The residue was chromatographed on silica gel (heptanes/DCM) togive 2.5 g (29%) of 2-amino-3-benzyl-5-phenylpyrazine.

Synthesis of 2-amino-3-phenylpropanenitrile hydrochloride. A roundbottomed flask was charged with 65 g (0.624 mol) of sodiumhydrogensulfite and 150 mL of water. A solution of 75 g (0.624 mol) ofphenylacetaldehyde in 150 mL of THF was added dropwise. After stirringfor 20 min, 37 mL of 14 M ammonium hydroxide was added in one portion,and the mixture heated to 60° C. for 60 min. After cooling to 0° C., themix was diluted with 150 mL of water, and a solution of sodium cyanide(27.5 g, 0.560 mol) in 100 mL of water added dropwise keeping internaltemperature below 10° C. Upon addition, the mixture was heated to 30° C.for 2 hrs and extracted with ether. After drying with sodium sulfate,all volatiles were evaporated, and the residue dissolved in 3.5 L ofether and treated with 400 mL of 3.3 M ethanolic HCl. The resultingprecipitate was filtered and dried in vacuum to give 55 g (60%) ofproduct.

Synthesis of 3-(furan-2-yl)-2-oxopropanoic acid. To a 100 mL flask,3-(furan-2-yl)-2-oxopropanoate (940 mg) along with 23 mL cold 6N NaOHwas added. The insoluble mixture was stirred in a 90° C. bath for 5 minuntil dissolved. Cold 1N HCl was added until solution was acidic (approx120 mL). Solution was extracted 2×50 mL EtOAc. Combined organic layerswere washed with 40 mL brine and dried with Na₂SO₄. Solution wasevaporated to yield 540 mg brown solid. Solid was further purified byreversed-phase high-performance liquid chromatography (HPLC) rampingfrom 97% aqueous trifluoroacetic acid (TFA) to acetonitrile (ACN).

Synthesis of ethyl 3-(furan-2-yl)-2-oxopropanoate. To a 500 mL flaskcontaining the mixture of isomers (E/Z)-ethyl2-formamido-3-(furan-2-yl)acrylate (5.0 g), a chilled solution of 220 mL1.4M (5%) HCl in 50/50 ethanol/water was added. After 5 hrs, thereaction was partitioned between 200 mL of EtOAc and 30 mL brine. Theaqueous layer was extracted 2×50 mL EtOAc. Combined organic layers werewashed with 1×50 mL water, and 1×50 mL brine and dried over Na₂SO₄.Organic layers were co-evaporated with 26 g celite and eluted over 80 gsilica gold ramping from heptane to EtOAc. The appropriate combinedfractions were evaporated to yield 2.1 g.

Synthesis of (E/Z)-ethyl 2-formamido-3-(furan-2-yl)acrylate. To a 500 mLflask, 50 mL diethyl ether, Cu₂O (320 mg), and furyl aldehyde (5.2 mL)was added. The flasked was cooled in an ice bath, and ethyl2-isocyanoacetate (5.3 mL) added. After 1.5 hrs, potassium tert-butoxide(5 g) was added to the reaction. After 4 hrs, the heterogeneous reactionwas filtered. 60 mL 30% citric acid and 20 mL EtOAc was added andstirred for 10 min. Aqueous layer was extracted with 50 mL EtOAc.Combined organic layers were dried over anhydrous sodium sulfate. EtOAclayers were co-evaporated with 24 g celite and eluted over 80 g silicagold ramping from heptane to EtOAc. Yellow syrup was used withoutfurther purification.

Synthesis of 2-oxo-3-(thiophen-2-yl)propanoic acid. To a 250 mL flask,(E/Z)-5-(thiophen-2-ylmethylene)imidazolidine-2,4-dione (5.0 g) and 100mL of cold 6N NaOH were added. The mixture was heated to 100° C. for 1hr. Concentrated HCl was added to the cooled solution until acidic(pH=1). The mixture was extracted 8×50 mL diethylether. The combinedether layers were washed with 50 mL brine, dried over Na₂SO₄ andevaporated to yield 3.36 g solid. Sample was further purified byrecrystallization with α,α,α-trifluorotoluene to yield 1.63 g.

Synthesis of (E/Z)-5-(thiophen-2-ylmethylene)imidazolidine-2,4-dione. Toa 250 mL flask, hydantoin (9.8 g) and thiophene-2-carbaldehyde (10 g)were added. To the mixture was dripped piperidine (9.6 mL). The mixturewas heated to 100° C. for 1 hr and then poured into 300 mL of 1N HCl.The solid was filtered, washed with water and dried in vacuo to yield4.9 g solid.

Steps 1—To a microwave vial (10 mL), the appropriatephenylpyrazin-2-amine (100 mg), the appropriate pyruvic acid (2Equivalents), DCM (1 mL), and 1,1,1-trifluoroethanol (1 mL) were heatedwith stirring for 30 min at 80° C. Reaction was co-adsorbed on 2 gramsof celite, and solvents removed in vacuo. The celite was loaded on 24 gof spherical silica gel and eluted with a ramp of heptanes toethylacetate. Appropriate fractions were combined and evaporated.

Step 2—The material isolated in step 1 dissolved in THF (0.5 mL) waschilled in an ice bath. Acetic anhydride (25 μL), dimethylaminopyridine(8.5 mg), and triethylamine (25 μL) were added. After 2 hrs, themajority of THF was removed in vacuo. The product was precipitated withan aqueous solution of 30% citric acid (2 mL). The solid was washed withwater (2 mL) and then dissolved in 3 mL DCM. The DCM was washed 1×2 mLwater followed by 1×2 mL brine. The DCM layer was co-adsorbed on 2 gramsof celite, and solvent removed in vacuo. The celite was loaded on 12 gof spherical silica gel and eluted with a ramp of heptanes to DCM.Appropriate fractions were combined and evaporated.

Step 3—The material from step 2 dissolved in DCM (1 mL) was chilled inan ice bath. To the solution, methanol (0.5 mL) and sodium borohydridesolution in diglyme (325 μL of 0.5 M) were added. After 2 hrs, aceticacid (10 μL) was added, and the solution quickly partitioned between anaqueous solution of 30% citric acid (1 mL) and DCM (2 mL). The DCM layerwas co-adsorbed on 1 gram of celite, and solvent removed in vacuo. Thecelite was loaded on 4 g of spherical silica gel and eluted with a rampof DCM to EtOAc. Appropriate fractions were combined and evaporated.

Step 4 (only if R″═OAc)—The material in step 3 was dissolved in THF (200μL) and chilled in an ice bath. 1 equivalent of 1.35 M potassiummethoxide in THF was added to the solution. After 30 min, the reactionwas partitioned between DCM (1 mL) and 30% citric acid (1 mL). The DCMlayer was co-adsorbed on 0.5 g celite, and solvent removed in vacuo. Thecelite was loaded on 4 g of spherical silica gel and eluted with a rampof DCM to EtOAc. Appropriate fractions were combined and evaporated.

Method D: (the following compounds can be synthesized by method D:compounds PBI-3899, PBI-3900, PBI-3925, PBI-3933, PBI-3946)—In general,an aminopyrazine was condensed with 2 equivalents of a 2-oxoacid underan atmosphere of hydrogen in the presence of palladium catalyst. Thealpha-amino acid produced was purified and subsequently activated forintramolecular condensation giving rise to the correspondingimidazopyrazinone.

Example 5 Synthesis of8-benzyl-6-(4-hydroxyphenyl)-2-propylimidazo[1,2-a]pyrazin-3(7H)-one

2-((3-benzyl-5-(4-hydroxyphenyl)pyrazin-2-yl)amino)pentanoic acid.4-(5-amino-6-benzylpyrazin-2-yl)phenol (100 mg, 0.36 mmol) was mixedwith 2-Oxovaleric acid (84 mg, 0.72 mmol) in ethanol (20 mL). Pd/C (10%Palladium in active carbon, 40 mg) was added, and the reaction mixtureheated to 65° C. Air was bubbled out by N₂ gas, and a hydrogen balloonapplied to the reaction flask. The reaction was continuously stirred for4 hrs. After cooling down, it was filtered, and the resulting solutionpurified by flash chromatography (eluting solvent: 50% EtOAc inheptanes) to give the product as a yellow powder (70 mg, 52%). ¹H NMR(300 MHz, CD₂Cl₂, δ): 8.31 (s, 1H), 7.82 (d, 2H, J=9.0 Hz), 7.31 (m,5H), 6.92 (d, 2H, J=9.0 Hz), 5.34 (s, 2H), 4.20 (m, 1H), 1.10 (m, 2H),0.98 (m, 2H), 0.87 (t, 3H); MS (ESI) m/z 378.3 (M+1).

8-benzyl-6-(4-hydroxyphenyl)-2-propylimidazo[1,2-a]pyrazin-3(7H)-one.2-((3-benzyl-5-(4-hydroxyphenyl)pyrazin-2-yl)amino)pentanoic acid (49mg, 0.13 mmol) was dissolved in DCM (10 mL). Pyridine (0.5 mL) was addedfollowed by N,N′-Dicyclohexylcarbodiimide (54 mg, 0.26 mmol). Thereaction mixture was slowly stirred at room temperature for 1 hr. Thesolvent was evaporated, and the residue purified by flash chromatography(eluting solvent: EtOAc to DCM to 10% methanol in DCM) to give theproduct as a yellow powder (40 mg, 86%). NMR (300 MHz, CD₃OD, δ): 7.35(m, 8H), 6.88 (d, J=9.0 Hz, 2H), 4.40 (s, 2H), 2.81 (t, J=7.5 Hz, 2H),1.81 (m, 2H), 1.02 (t, J=7.5 Hz, 3H); MS (ESI) m/z 359.0.

Example 6 Synthesis of8-benzyl-2-butyl-6-(4-hydroxyphenyl)imidazo[1,2-a]pyrazin-3(7H)-one

2-O-benzyl-5-(4-hydroxyphenyl)pyrazin-2-yl)amino)hexanoic acid.4-(5-amino-6-benzylpyrazin-2-yl)phenol (200 mg, 0.72 mmol) was mixedwith 2-Ketohexanoic acid sodium salt (220 mg, 1.44 mmol) in ethanol (20mL). Pd/C (10% Palladium in active carbon, 100 mg) was added with a fewdrops of acetic acid, and the reaction mixture heated to 65° C. Air wasbubbled out by N₂ gas, and a hydrogen balloon applied to the reactionflask. The reaction was continuously stirred for 4 hrs. After coolingdown, it was filtered and the resulting solution was purified by flashchromatography (eluting solvent: 50% EtOAc in heptanes) to give theproduct as a yellow powder (130 mg, 46%). MS (ESI): m/z 392.2 (M+1).

8-benzyl-2-butyl-6-(4-hydroxyphenyl)imidazo[1,2-a]pyrazin-3(7H)-one.2-((3-benzyl-5-(4-hydroxyphenyl)pyrazin-2-yl)amino)hexanoic acid (130mg, 0.33 mmol) was dissolved in DCM (10 mL). Pyridine (0.5 mL) was addedfollowed by N,N′-Dicyclohexylcarbodiimide (137 mg, 0.67 mmol). Thereaction mixture was slowly stirred at room temperature for 1 hr. Thesolvent was evaporated, and the residue purified by flash chromatography(eluting solvent: EtOAc to DCM to 10% methanol in DCM) to give theproduct as a yellow powder (110 mg, 89%). ¹H NMR (300 MHz, CD₃OD, δ):7.30 (m, 8H), 6.88 (d, 2H), 4.40 (s, 2H), 2.84 (t, 2H), 1.77 (m, 2H),1.51 (m, 2H), 0.89 (m, 3H); MS (ESI) m/z 374.3 (M+1).

Example 7 Synthesis of8-benzyl-2-ethyl-6-phenylimidazo[1,2-a]pyrazin-3(7H)-one (PBI-3925)

2-((3-benzyl-5-phenylpyrazin-2-yl)amino)butanoic acid3-benzyl-5-phenylpyrazin-2-amine (200 mg, 0.77 mmol) was mixed with2-Oxobutyric acid (157 mg, 1.54 mmol) in ethanol (20 mL). Pd/C (10%Palladium in active carbon, 100 mg) was added, and the reaction mixtureheated to 65° C. Air was bubbled out by N₂ gas, and a hydrogen balloonapplied to the reaction flask. The reaction was continuously stirred for4 hrs. After cooling down, it was filtered, and the resulting solutionpurified by flash chromatography (eluting solvent: 50% EtOAc inheptanes) to give the product as a yellow powder (90 mg, 34%). NMR (300MHz, CD₂Cl₂, δ): 7.72 (s, 1H), 7.32-7.48 (m, 10H), 4.46 (s, 2H), 4.20(m, 2H), 2.25 (q, 2H), 0.99 (t, 3H); MS (ESI): m/z 348.3 (M+1).

2-((3-benzyl-5-phenylpyrazin-2-yl)amino)butanoic acid was dissolved inDCM (10 mL). Pyridine (0.5 mL) was added followed byN,N′-Dicyclohexylcarbodiimide (137 mg, 0.67 mmol). The reaction mixturewas slowly stirred at room temperature for 1 hr. The solvent wasevaporated, and the residue purified by flash chromatography (elutingsolvent: EtOAc to DCM to 10% methanol in DCM) to give the product as ayellow powder (110 mg, 89%). ¹H NMR (300 MHz, CD₃OD, δ): 7.26 (m, 3H),6.84-7:07 (m, 8H), 4.03 (s, 2H), 2.47 (q, J=9.0 Hz, 2H), 0.96 (t, J=9.0Hz, 3H); MS (ESI): m/z 330.2 (M+1).

Example 8 Synthesis of8-benzyl-6-phenyl-2-(3,3,3-trifluoropropyl)imidazo[1,2-a]pyrazin-3(7H)-one

5,5,5-trifluoro-2-oxopentanoic acid. Ethyl 4,4,4-trifluorobutyrate (1 g,5.88 mmol) and diethyl oxalate (3.87 g, 26.5 mmol) was dissolved inethanol. Sodium ethoxide (21% in ethanol, 2.09 g) was added to thesolution, and the reaction mixture stirred for 0.5 hrs. Solvent wasdistilled, and the residue extracted with EtOAc/water. The organiclayers were collected and dried over sodium sulfate. After filtration,solvent was removed to give a clear liquid. MS (ESI): m/z 269.1 (M−1).The liquid was then dissolved in 3N HCl (20 mL), and the reactionmixture refluxed for 4 hrs. After cooling down, the reaction mixture wasextracted with EtOAc. The organic layers were collected and dried oversodium sulfate. After filtration, solvent was removed, and the residueused directly in the next step. MS (ESI): m/z 169.7 (M−1).

5,5,5-trifluoro-2-((3-benzyl-5-phenylpyrazin-2-yl)amino)butanoic acid.3-benzyl-5-phenylpyrazin-2-amine (240 mg, 0.92 mmol) was mixed with5,5,5-trifluoro-2-oxopentanoic acid (150 mg, 0.88 mmol) in ethanol (20mL). Pd/C (10% Palladium in active carbon, 100 mg) was added, and thereaction mixture heated to 65° C. Air was bubbled out by N₂ gas, and ahydrogen balloon applied to the reaction flask. The reaction wascontinuously stirred for 4 hrs. After cooling down, it was filtered, andthe resulting solution purified by flash chromatography (elutingsolvent: 50% EtOAc in heptanes) to give the product as a yellow powder(200 mg, 54%). ¹H NMR (300 MHz, CD₂Cl₂, δ): 11.45 (s, 1H), 10.20 (s,1H), 7.94 (s, 1H), 7.34 (m, 10H), 5.34 (s, 2H), 3.96-4.23 (m, 2H),3.02-3.28 (m, 2H); ¹H NMR: −76.3; MS (ESI): m/z 416.1 (M+1).

Coelenterazine (R₁═H, R₂═—CH₂CH₂CF₃).5,5,5-trifluoro-2-((3-benzyl-5-phenylpyrazin-2-yDamino)butanoic acid(100 mg, 0.24 mmol) was dissolved in DCM (10 mL). Pyridine (0.5 mL) wasadded followed by N,N′-Dicyclohexylcarbodiimide (100 mg, 0.48 mmol). Thereaction mixture was slowly stirred at room temperature for 1 hr. Thesolvent was evaporated, and the residue purified by flash chromatography(eluting solvent: EtOAc to DCM to 10% methanol in DCM) to give theproduct as a yellow powder (80 mg, 87%). ¹H NMR (300 MHz, CD2Cl₂, δ):7.36 (m, 11H), 3.43 (s, 2H), 1.60-1.92 (m, 4H); ¹H NMR: 67.4 (t, J=18Hz); MS (ESI): m/z 398.2 (M+1).

Example 9 Synthesis of8-benzyl-2-(furan-2-ylmethyl)-6-phenylimidazo[1,2-a]pyrazin-3(7H)-one(PBI-3939)

8-benzyl-2-(furan-2-ylmethyl)-6-phenylimidazo[1,2-a]pyrazin-3(7H)-one:Synthesized from method C using 3-(furan-2-yl)-2-oxopropanoic acid and3-benzyl-5-phenylpyrazin-2-amine as starting materials. ¹H NMR (300 MHz,dmso) δ 8.88 (s, 1H), 8.02 (d, J=7.9, 2H), 7.61-7.38 (m, 6H), 7.37-7.14(m, 3H), 6.38 (s, 1H), 6.26 (d, J=3.2, 1H), 4.64 (s, 3H), 4.40 (s, 3H);exact mass calculated for C₂₄H₂₀N₃O₂ ⁺ m/z+ 382.16, found m/z+ 382.

Example 10 Synthesis of8-benzyl-6-phenyl-2-(thiophen-2-ylmethyl)imidazo[1,2-a]pyrazin-3(7H)-one(PBI-3889)

8-benzyl-6-phenyl-2-(thiophen-2-ylmethyl)imidazo[1,2-a]pyrazin-3(7H)-one:Synthesized from method C using 2-oxo-3-(thiophen-2-yl)propanoic acidand 3-benzyl-5-phenylpyrazin-2-amine as starting materials. ¹H NMR (300MHz, dmso) δ 8.85 (s, 1H), 7.99 (d, J=6.8, 2H), 7.63-7.02 (m, 10H), 6.94(dd, J=3.5, 5.1, 1H), 4.62 (s, 2H), 4.58 (s, 2H), 2.69 (contaminate);exact mass calculated for C₂₄H₂₀N₃OS₊ m/z+ 398.13, found m/z+ 398.

Example 11 Synthesis of8-cyclopropyl-2-(4-hydroxybenzyl)-6-phenylimidazo[1,2-a]pyrazin-3(7H)-one(PBI-3897)

8-cyclopropyl-2-(4-hydroxybenzyl)-6-phenylimidazo[1,2-a]pyrazin-3(7H)-one:Synthesized using method A with 3-cyclopropyl-5-phenylpyrazin-2-amineand 3-(4-hydroxyphenyl)-2-oxopropanoic acid as starting materials. Exactmass calculated for C₂₂H₁₈N₃O₂ ⁻ m/z− 356.14, found m/z− 356.

Example 12 Synthesis of8-benzyl-2-methyl-6-phenylimidazo[1,2-a]pyrazin-3(7H)-one (PBI-3932)

8-benzyl-2-methyl-6-phenylimidazo[1,2-a]pyrazin-3(7H)-one: Synthesizedusing method B with 1,1-dimethoxypropan-2-one and3-benzyl-5-phenylpyrazin-2-amine as starting materials. Exact masscalculated for C₂₀H₁₈N₃₀ ⁺ m/z+ 316.14, found m/z+ 316.

Example 13 Synthesis of2-(4-hydroxybenzyl)-8-methyl-6-phenylimidazo[1,2-a]pyrazin-3(7H)-one(PBI-3896)

2-(4-hydroxybenzyl)-8-methyl-6-phenylimidazo[1,2-a]pyrazin-3(7H)-one:Synthesized using method A with 3-methyl-5-phenylpyrazin-2-amine and3-(4-hydroxyphenyl)-2-oxopropanoic acid as starting materials. ¹H NMR(300 MHz, dmso) δ 8.84 (s, 1H), 8.00 (d, J=7.6, 2H), 7.47 (dd, J=8.6,16.2, 3H), 7.17 (d, J=7.3, 2H), 6.69 (d, J=8.4, 2H), 6.26 (s, 4H), 4.17(s, 2H), 2.86 (s, 3H), 2.48 (s, 1H).

Example 14 Synthesis of8-benzyl-2-(4-hydroxybenzyl)-6-phenylimidazo[1,2-a]pyrazin-3(7H)-one(PBI-3840)

8-benzyl-2-(4-hydroxybenzyl)-6-phenylimidazo[1,2-a]pyrazin-3(7H)-one:Synthesized using method A with 3-(4-hydroxyphenyl)-2-oxopropanoic acidand 3-benzyl-5-phenylpyrazin-2-amine as starting materials. Exact masscalculated for C₂₆H₂₂N₃O₂ ⁺ m/z+ 408.17, found m/z+ 408.

Example 15 Synthesis of Protected Coelenterazine (Stabilized) (PBI-4377)

To a mixture of PBI-3939, potassium carbonate (1.1 equiv) and potassiumiodide (1.1 equiv) in dimethylformamide (DMF), under an argonatmosphere, was added one equivalent of chloromethyl pivalate at roomtemperature. Reaction progress was monitored by thin layerchromatography, and upon completion, the reaction mixture was cooled inan ice bath for several minutes before addition of a volume of waterequal to the reaction volume. The resulting mixture was extracted with asuitable organic solvent (e.g., EtOAc), and the extract was concentratedto give the crude product. The material was further purified bychromatography over silica gel.

Example 16 Synthesis of8-benzyl-2-((1-methyl-1H-imidazol-2-yl)methyl)-6-phenylimidazo[1,2-a]pyrazin-3(7H)-one(PBI-4525),8-benzyl-6-(4-hydroxyphenyl)-2-((1-methyl-1H-imidazol-2-yl)methyl)-6-phenylimidazo[1,2-a]pyrazin-3(7H)-one(PBI-4540) and2-((1H-imidazol-2-yl)methyl)-8-benzyl-6-phenylimidazo[1,2-a]pyrazin-3(7H)-one(PBI-4541)

To a flask containing 10 mmol of 2-methyl imidazole derivative 1 or 2under an argon atmosphere, 20 mL of dry THF was added, and the solutionwas cooled in a dry ice/acetone bath to approximately −78° C. To thecold mixture, 9.3 mmol of a solution of n-butyllithium (2.46 M inHexanes) was added dropwise over several minutes. The resulting solutionwas stirred at approximately −78° C. for 30 min, and 6.7 mmol ofcompound 3 was added via syringe. The reaction mixture was stirred for 3hrs and quenched with the addition of 20 mL saturated ammonium chloridesolution and 20 mL of saturated sodium bicarbonate solution. The coldbath was removed, and after warming to room temperature, the mixture wasextracted with 3×100 mL of EtOAc. The combined extracts were dried(MgSO₄), concentrated in vacuo, and the crude compounds 4 or 5 werepurified by column chromatography using silica gel (EtOAc/Heptane).

A microwave vial was charged with 100 mg (1 eq) of compound 6 or 7 and 2equivalents of compound 8 or 9. To the mixture, 4.5 mL of ethanol and0.25 mL of concentrated HCl was added. The reaction mixture was heatedin a microwave at 100° C. for 1.5 hr. The resulting mixture was added to50 mL of EtOAc and washed sequentially with 20 mL of saturated sodiumbicarbonate solution and 20 mL of brine. The organic phase wasconcentrated in vacuo, and the residue purified by column chromatographyusing silica gel (methanol/dichloromethane) to give compounds 10-12.

Example 17 Stability and Auto-Luminescence Characterization of NovelCoelenterazines

The stability and auto-luminescence characterization of the novelcoelenterazines PBI-3939, PBI-3889, PBI-3945, PBI-4002, or PBI-3896 weredetermined. Higher stability and less auto-luminescence is an attractivetechnical feature in a substrate/reagent.

To determine stability, 20 μM of novel coelenterazines PBI-3939,PBI-3889, PBI-3945, PBI-4002, or PBI-3896, 30 μM native coelenterazine,or 22 μM of known coelenterazine-h or known coelenterazine-hh, wereplaced in a reporter reagent buffer containing 50 mM CDTA, 150 mM KCl,50 mM DTT, 35 mM thiourea, 1% TERGITOL® NP-9 (v/v), and 0.1% MAZU> DF204. Replicate samples were incubated at room temperature (i.e., 22-24°C.) for various lengths of time and then transferred to −70° C. Afterall the samples were collected and frozen, they were thawed and mixedwith 10 μL of bacterial cell lysate containing the OgLuc variant IV in40 μL of DMEM without phenol red+0.1% PRIONEX®. The luminescence of thesample was read at 5 min after IV addition.

“T₉₀” indicates the amount of time for the luminescent signal to decayby 10% (i.e., loss in activity by 10%) at ambient temperature, i.e., 22°C. The rate of decay of the luminescent signal (“T₉₀”) was determinedfrom the slope of the linear fit of the data plotted as In RLU vs. time,which was calculated from the following equation: t=ln (A/A₀)÷(−k),where A=intensity at time t, A₀=intensity at time 0, and k=the rate ofdecay. As shown in Table 1, the T₉₀ values for known coelenterazines-hand -hh, novel coelenterazines PBI-3939, PBI-3889, PBI-3945, PBI-4002,and PBI-3896 were higher than for native coelenterazine, indicating thatthese coelenterazines were more stable compounds than nativecoelenterazine.

To determine the auto-luminescence characterization, HEK293 cells weregrown overnight at 15,000 cells per well in DMEM+10% FBS+pyruvate. Mediawas removed and replaced with 20 μM each of the novel coelenterazinesshown in FIG. 2, i.e., PBI-3939, PBI-3889, PBI-3945, PBI-4002, PBI-3841,PBI-3897, PBI-3896, PBI-3925, PBI-3894, PBI-3932, and PBI-3840, nativecoelenterazine and known coelenterazines, coelenterazine-h andcoelenterazine-hh, diluted into CO₂ independent media plus 10% FBS.Luminescence was measured shortly after substrate addition on theGLOMAX® Luminometer (1 sec/well). Background luminescence was 154±15RLU. Table 1 shows the auto-luminescence characterization normalized tonative coelenterazine (“Autolum (norm to coel)”). While coelenterazine-hhad more auto-luminescence than native coelenterazine, all of the othercoelenterazines tested had less auto-luminescence.

TABLE 1 Stability Experiments and Autoluminescence Characterization ofIV with Various Coelenterazines. Stability (pH 8) Autolum Substrate ID(T₉₀ in hrs) (norm to coel) Coel 1.7 1 Coel h 2.1 1.2 Coel hh 2.0 0.33939 4.1 0.2 3889 2.9 0.2 3945 3.3 0.5 4002 3.5 0.6 3841 0.1 3897 0.13896 2.8 0.1 3894 0.2 3932 0.1 3840 0.2 3925 0.2

Example 18 Toxicity of Novel Coelenterazines

The toxicity of the novel coelenterazines were investigated in HEK293cells. HEK293 cells were grown overnight at 15,000/well in DMEM+10%FBS+pyruvate. The media was removed and replaced with the novelcoelenterazine compounds (or DMSO control) diluted into CO₂ independentmedia plus 10% FBS. Cell viability was measured 24 hrs after compoundaddition using CELLTITER-GLO® assay reagent (Promega Corp.) according tothe manufacturer's instructions, and luminescence was measured on theGLOMAX® Luminometer (1 sec/well). Table 2 shows the toxicity of thenative coelenterazine, known coelenterazine-h and coelenterazine-hh, andthe novel coelenterazines PBI-3939, PBI-3889, PBI-3841, PBI-3897,PBI-3945, PBI-4002, and PBI-3840 in HEK293 cells. With the exception ofPBI-3840, the novel coelenterazines had at least the same toxicity ascoelenterazine-hh. Some of the novel coelenterazines had the sametoxicity as native coelenterazine and coelenterazine-h.

TABLE 2 Toxicity of Various Coelenterazines in HEK293 Cells Based onCELLTITER-GLO ® Viability (normalized to vehicle Substrate (DMSO)control) (%) Native coelenterazine 100 Coelenterazine h 100Coelenterazine hh 87 PBI-3939 89 PBI-3889 90 PBI-3841 100 PBI-3897 100PBI-3945 100 PBI-4002 100 PBI-3840 60

Example 19 Km of PBI-3939

To determine the Km of PBI-3939, the OgLuc variant L27V (described inExample 26) was purified via HALOTAG® fusion using the HALOTAG® ProteinPurification System according to the manufacturer's instructions anddiluted in DMEM without Phenol Red and 0.1% PRIONEX®. 50 μL assay buffer(100 mM MES pH 6, 35 mM Thiourea, 0.5% TERGITOL® NP-9 (v/v), 1 mM CDTA,2 mM DTT and 150 mM KCl) with varying amounts of PBI-3939 was added to50 μL diluted enzyme (approximately 20 μM final enzyme concentration),and luminescence measured at 3 min at 22° C. As the data in FIG. 3demonstrates, the Km of PBI-3939 is approximately 10 μM.

Example 20 Characterization of compounds PBI-4525, PBI-4540 and PBI-4541

Compounds PBI-4525, PBI-4540 and PBI-4541 were screened for theirability to detect luminescence. For analysis, 20 μM of each compound wasadded to assay buffer (100 mM MES pH 6, 35 mM Thiourea, 0.5% TERGITOL®NP-9 (v/v), 1 mM CDTA, 2 mM DTT and 150 mM KCl) which was adjusted to pH7 with 100 mM HEPES pH 7 to create an assay reagent. The assay reagentwas then mixed with 36 μM purified L27V02 enzyme (described in Example25B), in DMEM without Phenol Red and 0.1% PRIONEX®. As a control, assaybuffer with 20 μM PBI-3939 or PBI-4528 was used. Luminescence wasmeasured as previously described 3 min after the assay reagent was addedto the enzyme mixture. Table 3 demonstrates that compounds PBI-4525,PBI-4540 and PBI-4541 can be used to detect luminescence from acoelenterazine-utilizing luciferase.

TABLE 3 cmpd RLU +/− 4525 20,655 1,006 4528 202,080 5,688 3939 9,808,880307,565 4540 909 7 4541 5,676 80

Example 21 OgLuc Pattern Sequence

Enzyme families, including different classes of luciferases, can berecognized by having common three-dimensional structures and definedcatalytic activity. Because enzyme families share evolutionary historieswith other enzyme families, they will also exhibit similarities in theirthree-dimensional structures. Through various means of structural andfunctional analysis, the inventors have determined that OgLuc, as arepresentative of decapod luciferases, has a strikingly similarthree-dimensional structure to Fatty Acid Binding Proteins (FABPs),indicating commonality of evolutionary history. Thus, decapod luciferasemay be defined as having a characteristic three-dimensional structuresimilar to FABPs and utilizing coelenterazine as a substrate to catalyzethe emission of luminescence. Other luciferases, e.g., fireflyluciferase, Renilla luciferase, bacterial luciferase, and so forth, haveclearly distinct three-dimensional structures, indicating that theybelong to different enzyme families and do not share evolutionaryhistories. Dinoflagellate luciferase has a three-dimensional structureexhibiting some similarities to FABPs, suggesting a shared evolutionaryhistory, but does not utilize coelenterazine as a substrate, and thusdoes not belong to the same enzyme family as decapod luciferases.

Because amino acid sequences are not as well conserved asthree-dimensional structures, defining enzyme families based only onsequence comparisons can be difficult. For example, even though FABPsall have a characteristic barrel-shaped three-dimensional shape,comparisons of their amino acid sequences often reveal very low levelsof sequence identity. Nonetheless, sequence identity can be used todemonstrate commonality of three-dimensional structures. Two proteinswill have analogous three-dimensional structures if their amino acidsequences can be aligned to reveal >30% sequence identity,preferably >40% sequence identity, and most preferably >50% sequenceidentity (Chothia and Lesk, EMBO J. 5(4):823-826 (1986); Tramontano,Genomics, 4:402-405 (2003)). Thus, a protein is a decapod luciferase if,upon alignment of its amino acid sequence with the sequence of OgLuc,the sequence identity is >30%, preferably >40%, and mostpreferably >50%, and the protein can utilize coelenterazine as asubstrate to catalyze the emission of luminescence.

Because of structural constraints necessary to sustain thecharacteristic three-dimensional structure of an enzyme family, someportions of the amino acid sequences in an enzyme family exhibit greateramounts of conservation (i.e., a greater level of sequence identity).Thus, these conserved regions can serve as further evidence of a commonthree-dimensional structure shared between two proteins. A conservedsequence pattern, also called a signature, motif, or fingerprint, can begenerated by manual or computational methods that are known in the art.Patterns can be found in public databases such as PROSITE(http://expasy.org/prosite; Sigrist et al., Nucleic Acids Res. 38(suppl1):D161-D166 (2010)).

For example, a pattern of conserved amino acids can be found uponexamination of a large number of known FABPs. PROSITE (Release 20.67, of5 Oct. 2010) contains an FABP pattern (accession number PS00214, createdApril-1990, data updated April-2006). This FABP pattern spans 18 aminoacid positions and is defined as:

(VI) (SEQ ID NO: 329) [GSAIVK]-{FE}-[FYW]-x-[LIVMF]-x-x-{K}-x-[NHG]-[FY]-[DE]-x-[LIVMFY]-[LIVM]-{N}-{G}-[LIVMAKR],

where:

the standard IUPAC one-letter codes for the amino acids are used.

the symbol ‘x’ is used for a position where any amino acid is accepted.

alternative amino acids at a site are indicated by listing the aminoacids between square parentheses ‘[ ]’ (for example: [ALT] representsthe possibility of an Ala, Leu, or Thr at the position).

the absence of particular amino acids at a site is indicated by curlybrackets ‘{ }’ (for example: {AM} represents any amino acid at aposition except Ala and Met).

each sequence position (or element in the pattern) is separated from itsneighbor by ‘-’.

each sequence position is referred to as a “pattern position”, forexample the [GSAIVK] would be considered pattern position 1 of Formula(VI), {FE} is considered pattern position 2 of Formula (VI), etc.

Although a conserved sequence pattern results from a common underlyingthree-dimensional structure, some changes to the sequence pattern may beallowed without disruption to the three-dimensional structure. Forexample, for some members of the FABP family, differences are found atfour sites in the PROSITE pattern. These additional members of the FABPfamily include five proteins listed in PROSITE as false negative hits,i.e., FABP protein family members not picked up by the FABP pattern(UniProt database accession numbers FBP12_HUMAN, FABP1_FASGI,FABP2_FASHE, FABPL_SCHBI, RET5_BOVIN) and one protein known to have anFABP fold (Protein Data Bank accession number 2A02). Although OgLucshares a closely similar three-dimensional structure with FABPs, thesequence patterns of the native and variant amino acid sequences alsodiffer slightly, having differences at 5 positions from the PROSITEpattern. In various embodiments, the pattern in OgLuc begins at aposition corresponding to position 8 of SEQ ID NO: 1. An amino acidsubstitution, deletion, or insertion the sequence pattern is counted asa difference.

Combining the sequence information from these additional FABPs and theOgLuc variants, an improved sequence pattern can be derived:

(VII) (SEQ ID NO: 330)[GSAIVK]-{FE}-[FYW]-x-[LIVMFSYQ]-x-x-{K}-x-[NHGK]-x-[DE]-x-[LIVMFY]-[LIVMWF]-x-{G}-[LIVMAKRG].

The sequence information used to derive this pattern is shown in Table4. Column 1 identifies the pattern position (listed N- to C-terminus;pattern length is 18 amino acids), and column 6 identifies thecorresponding sequence position in OgLuc (numbering according to SEQ IDNO: 1). Column 2 shows the PROSITE FABP pattern (Formula (VI)) elementfor each pattern position. Column 3 lists amino acids present in sixFABP family members that are not represented by the PROSITE FABPpattern. Column 4 lists amino acids present in OgLuc (SEQ ID NO: 1) orOgLuc variants that are not represented by the PROSITE pattern. Column 5lists the improved pattern (“OgLuc pattern”) (Formula (VII)) created bymerging pattern information from columns 2, 3, and 4. Column 7 lists theamino acids in OgLuc (SEQ ID NO: 1) corresponding to the PROSITE FABPpattern positions. Column 8 lists the amino acids found indinoflagellate luciferase sequences (8 different species) at positionscorresponding to the improved pattern (GenBank accession numbers2021262A, AAA68491, AAC36472, AAV35379, AAV35380, AAL40676, AAL40677,AAV35378, AAV35377, AAV35381, and Protein Data Bank accession number1VPR).

The improved pattern (Formula (VII)) serves as an indication (i.e., afingerprint) of the three-dimensional protein structure shared betweenFABPs and OgLuc. However, strict agreement with this pattern is notneeded to indicate commonality of the three-dimensional structure. Fromthe examples given here, a common three-dimensional structure may existeven with as many as 5 changes in the pattern. Also, for example,although the dinoflagellate luciferase has a similar three-dimensionalstructure to FABPs and OgLuc, it has 4 differences from the improvedpattern.

Thus, although a protein may be recognized as being a decapod luciferasebased on sequence similarity and utilization of coelenterazine forluminescence, it can be further recognized by also having the improvedsequence pattern. Specifically, a protein is a decapod luciferase if,upon alignment of its amino acid sequence with SEQ ID NO: 1 or variantsthereof, the sequence identity is >30%, preferably >40%, and mostpreferably >50%, and the protein can utilize coelenterazine as asubstrate to catalyze the emission of luminescence, and the amino acidsequence beginning at the position corresponding to position 8 of SEQ IDNO: 1 is:

(VII) (SEQ ID NO: 330)[GSAIVK]-{FE}-[FYW]-x-[LIVMFSYQ]-x-x-{K}-x-[NHGK]-x-[DE]-x-[LIVMFY]-[LIVMWF]-x-{G}-[LIVMAKRG],

with no more than 5 differences, or more preferably no more than 4, 3,2, or 1 difference, or most preferably no differences, wherein thedifferences occur in positions corresponding to pattern position 1, 2,3, 5, 8, 10, 12, 14, 15, 17, or 18 of Formula (VII) according to Table4. Differences may also include gaps or insertions between the patternpositions of Table 4.

TABLE 4 Protein sequence patterns PROSITE Pattern FABP pattern OtherOgLuc wt OgLuc OgLuc wt Dinofl. position PS00214 FABPs & variantsOgLuc pattern position sequence Luc  1 [GSAIVK] [GSAIVK]  8 G G(SEQ ID NO: 427) (SEQ ID NO: 579)  2 {FE} {FE}  9 D R  3 [FYW] [FYW] 10W W  4 x x 11 Q I  5 [LIVMF] [LIVMFSYQ] 12 Q T (SEQ ID NO: 590) SY Q(SEQ ID NO: 591)  6 x x 13 T [VI]  7 x x 14 A S  8 {K} {K} 15 G G  9 x x16 Y G 10 [NHG] K [NHGK] 17 N Q (SEQ ID NO: 580) 11 [FY] SILM {FY} X 18Q [AVTK] (SEQ ID (SEQ ID NO: 581) NO: 582) 12 [DE] [DE] 19 D [AE] 13 x x20 Q F 14 [LIVMFY] [LIVMFY] 21 V I (SEQ ID NO: 583) (SEQ ID NO: 584)[LIVM] [LIVMWF] 15 (SEQ ID NO: 585) W F (SEQ ID NO: 586) 22 L K 16 {K} Kx 23 E [EKTQ] (SEQ ID NO: 587) 17 {G} {G} 24 Q [AV] 18 [LIVMAKR] G[LIVMAKRG] 25 G [VI] (SEQ ED NO: 588) (SEQ ID NO: 589)

Example 22 Generation of OgLuc Variants

Experimental Details

Unless otherwise stated, further variants of a starting OgLuc variantsequence with random substitutions were generated using the error-prone,mutagenic PCR-based system GeneMorph II Random Mutagenesis Kit(Stratagene; Daughtery, PNAS USA, 97(5):2029 (2000)), according tomanufacturer's instructions, and NNK site saturation (Zheng et al.,Nucleic Acids Research, 32:e115 (2004)).

Further variants of a starting OgLuc variant having specific mutationswere generated using the oligo-based site-directed mutagenesis kitQuikChange Site-Directed Mutagenesis Kit (Stratagene; Kunkel, PNAS USA,82(2):488 (1985)), according to the manufacturer's instructions.

The resulting variants were constructed in the context of pF1K FLEXI®vector for T7 promoter-based expression (Promega Corp.). Alternatively,the resulting variants were constructed in the context of pF4Ag vector(a version of the commercially-available pF4A (Promega Corp.), whichcontained T7 and CMV promoters modified to contain an E. coliribosome-binding site with or without a C-terminal HALOTAG® (PromegaCorp.; referred herein as “HT7”) (Ohana et al., Protein Expression andPurification, 68:110-120 (2009)) to generate a fusion protein. Forexample, to obtain C1+A4E variants, NNK saturation mutagenesisexperiments were performed in a pF1K vector background. The C1+A4Elibrary was generated in a pF4Ag vector background with no HT7. TheQC27, QC27-9a, and IVY libraries were generated in a pF4Ag vectorbackground with a C-terminal HT7. The IV-based variants were generatedin a pF4Ag vector background without HT7. The resulting vectors wereused to transform KRX E. coli using techniques known in the art.

Generated OgLuc variants are named for the amino acid substitutionsidentified in the variant and/or for the E. coli clone that containedthe variant, e.g., FIG. 6A shows, among other results, that E. coliclone 16C5 has the substitution Q20R.

Screening Details

Resulting libraries were expressed in E. coli and primarily screenedwith a robotic system for OgLuc variants having increased light output(i.e., increased luminescence, increased brightness, or increased lightemission) or a change in relative specificity compared to thecorresponding starting OgLuc variant. The robotic primary screen wasconducted as follows: individual colonies from the generated librarywere used to inoculate minimal media in 96-well plates and grown at 37°C. for 17 to 20 hrs (“M1 culture”). The M1 culture was diluted 1:20 withfresh minimal media and grown at 37° C. for 17-20 hrs (“M2 culture”).The M2 culture was diluted 1:20 into induction media and grown 17-20 hrsat 25° C. with walk-away induction, i.e., autoinduction (Schagat et al.,“KRX Autoinduction Protocol: A Convenient Method for ProteinExpression.” Promega Notes, 98:16-18 (2008)). The induction mediacontained rhamnose and glucose when novel coelenterazines PBI-3841,PBI-3842, PBI-3857, PBI-3880, PBI-3881, PBI-3886, PBI-3887, PBI-3897,PBI-3896, or PBI-3894 were used as substrates in the primary screen. Theinduction media did not contain rhamnose or glucose when nativecoelenterazine, known coelenterazine-h, or novel coelenterazinesPBI-3840, PBI-3889, PBI-3899, or PBI-3900 were used as substrates in theprimary screen. The use of the different induction media was determinedbased on the luminescence generated between C1+A4E and the novelcoelenterazines, i.e., the induction media containing rhamnose andglucose were used with novel coelenterazines that generated lessluminescence with C1+A4E compared to the other novel coelenterazineswith C1+A4E.

Ten μL of induced cells were lysed using 60 μL lysis buffer containing300 mM HEPES pH 8.0, 300 mM thiourea, 0.3× Passive Lysis Buffer (“PLB”;Promega Corp. Cat. No. E194A), 0.3 mg/mL lysozyme, and 0.003 U/μL RQ1DNase and measured for luminescence with 50 μL assay buffer containing150 mM KCl, 1 mM CDTA, 10 mM DTT, 0.5% TERGITOL® NP-9 (v/v), and 20 μMof a native, known, or novel coelenterazine as a substrate. Luminescencemeasurements for each variant were taken 3 min after reagent additionand relative luminescence unit (RLU) values were normalized to anaverage of 8 control wells of the corresponding starting OgLuc variantfor each plate. Assay was completed on a TECAN® robotic system.

OgLuc variants of interest were sequenced using standard sequencingtechniques known in the art to identify any additional amino acidsubstitutions in each such variant. A secondary screen using anon-robotic (manual) system was performed on the variant clones ofinterest. The manual screen was conducted as follows: Variant cloneswere grown, in triplicate, in 96-well plates and expressed and assayedas described for the automated assay except the assay buffer was addedmanually with a multichannel pipette. For each variant, luminescence wasmeasured, averaged, and normalized to the corresponding starting OgLucvariant. Luminescence measurements were made using a TECAN® INFINITE®F500 luminometer.

Determining Change in Relative Specificity

Relative substrate specificity was determined by dividing theluminescence of a luciferase in the presence of a test coelenterazinesubstrate by the luminescence of the luciferase in the presence of areference coelenterazine substrate. For example, relative specificitywas determined by dividing the luminescence of a luciferase with a novelcoelenterazine of the present invention by the luminescence of theluciferase with a different coelenterazine (e.g., native or knowncoelenterazine, or a different novel coelenterazine of the presentinvention). The test coelenterazine substrate and the referencecoelenterazine substrate that were compared were considered a comparisonsubstrate pair for determining relative substrate specificity.

A change in relative substrate specificity was determined by dividingthe relative substrate specificity of a test luciferase using acomparison substrate pair by the relative substrate specificity of areference luciferase using the same comparison substrate pair. Forexample, a change in relative specificity was determined by dividing therelative substrate specificity of a test luciferase with a novelcoelenterazine of the present invention compared to a differentcoelenterazine (e.g., native or known coelenterazine or a differentnovel coelenterazine of the present invention), by the relativesubstrate specificity of a reference luciferase with the same novelcoelenterazine of the present invention compared to the same differentcoelenterazine used for the test luciferase.

The luminescence with one novel coelenterazine was compared to theluminescence with a different novel coelenterazine. The luminescencewith one native or known coelenterazine was compared to the luminescencewith another native or known coelenterazine. The luminescence with onenative or known coelenterazine was compared to the luminescence with anovel coelenterazine.

An increase in luminescence (RLUs) for the OgLuc variant compared to thecorresponding starting OgLuc template for novel coelenterazine and adecrease or no change in luminescence for a reference coelenterazine wasindicative of a change in relative specificity. A decrease inluminescence of an OgLuc variant for both the novel and referencecoelenterazines compared to the corresponding starting OgLuc, but theluminescence of the OgLuc variant with the novel coelenterazinedecreasing more, was also indicative of a change in relativespecificity. An increase in luminescence of the OgLuc variant comparedto the corresponding starting OgLuc for the novel and referencecoelenterazines indicated an improvement inactivity/stability/expression. If the luminescence of the OgLuc variantwith both the novel and the reference coelenterazines increased, but theincrease in luminescence with the novel coelenterazine was greater, itindicated an increase in relative specificity and an improvement inactivity/stability/expression of the OgLuc variant.

A. C1+A4E Variants

C1+A4E (SEQ ID NOs: 2 and 3), previously described in U.S. applicationSer. No. 12/773,002 (U.S. Published Application No. 2010/0281552), wasused as a primary starting sequence (i.e., the parental sequence) forgenerating additional, synthetic OgLuc variants. C1+A4E has thefollowing amino acid substitutions: A4E, Q11R, A33K, V44I, A54F, P115E,Q124K, Y138I, and N166R, relative to SEQ ID NO: 1. Luminescence ofC1+A4E containing bacterial lysates, using the novel coelenterazinesdescribed in Examples 1-14 (see FIG. 4 for examples) as substrates, wasmeasured as described previously and compared to the luminescence usingnative and known coelenterazines as substrates (FIGS. 5A-G). FIG. 5Ashows the luminescence of C1+A4E using native coelenterazine(“coelenterazine”), known PBI-3880, and novel coelenterazines PBI-3842,PBI-3857, PBI-3881, PBI-3882, PBI-3886, and PBI-3887 as substrates. Theluminescence measurements using known and novel coelenterazines werenormalized to the luminescence of C1+A4E using native coelenterazine andthe fold-decrease compared to native coelenterazine (FIG. 5B). FIGS.5C-E show the luminescence of C1+A4E using native coelenterazine andnovel coelenterazines PBI-3945, PBI-3894, and PBI-4002, respectively.FIG. 5F shows the luminescence of C1+A4E using native coelenterazine andnovel coelenterazines PBI-3840, PBI-3897, PBI-3889, PBI-3899, andPBI-3900. FIG. 5G shows the luminescence of C1+A4E using nativecoelenterazine, known coelenterazine PBI-3912 and novel coelenterazinesPBI-3913, PBI-3925, PBI-3939, PBI-3933, PBI-3932, PBI-3946, PBI-3841,and PBI-3896. The data indicates the C1+A4E variant can use each of thenovel coelenterazines as substrates.

C1+A4E variants were generated that had at least the amino acidsubstitutions identified in C1+A4E, unless otherwise indicated. Alibrary (Library 1) of 4400 variant clones of C1+A4E was generated byrandom mutagenesis as described previously and screened as describedpreviously for improvement in relative specificity change and/oractivity change, e.g., brightness. The variants were primarily screenedwith native coelenterazine, known coelenterazine-h, known PBI-3880, andnovel coelenterazines PBI-3840, PBI-3841, PBI-3842, PBI-3857, PBI-3881,PBI-3886, PBI-3887, PBI-3889, PBI-3897, and PBI-3900 as substrates. Inaddition, half of the variants were screened with novel coelenterazinesPBI-3896 and PBI-3894 as substrates. Plates containing variants havingknown mutations of interest identified from screening previous novelcompounds were selected. Variants that showed improvement (eitherrelative specificity change or activity change) for one or more of thenovel coelenterazines tested in the primary screen were isolated,sequenced, and screened in a secondary screen.

In the secondary manual screen, the variants were tested with knowncoelenterazines PBI-3912, coelenterazine-h, coelenterazine-hh, 2-methylcoelenterazine, and coelenterazine v; and novel coelenterazinesPBI-3840, PBI-3897, PBI-3889, PBI-3899, PBI-3900, PBI-3925, PBI-3944,PBI-3932, PBI-3945, PBI-3913, and PBI-3896 as substrates. FIGS. 6A-Dsummarize the average luminescence normalized to C1+A4E for the variants(“Clone”). FIGS. 6A-D summarize the substitutions in these variants (“AAsequence”), which had at least one of the following additional aminoacid substitutions: A14V, G15R, Q18L, Q20R, L22I, E23K, L27V, L27M,K33N, T39I, E49K, F54S, F54I, D55G, 156V, V58I, V58L, 159T, S66T, G67S,F68S, L72Q, M75K, I76N, F77T, F77C, K89E, I90V, I90T, L92H, H93R, M106K,Y109F, P113T, 1117F, T126R, V127A, L136M, D139G, P145L, S148T, C164S, orA169V.

Amino acid substitutions at position 54, 92, and 109 were of interest assubstitutions at these positions provided greater light output orimproved relative specificity, i.e., specificity away from nativecoelenterazine and towards at least one novel coelenterazine, as shownin FIGS. 6A-C. The amino acid substitution F54I in clone 29H7 providedgreater light output with native coelenterazine and several of the novelcoelenterazines. The amino acid substitution Q18L in clone 40H11, theamino acid substitution L92H in clone 04A12, and the amino acidsubstitution Y109F in clone 43F9 provided improved relative specificity.

Table 5 lists C1+A4E variants with an additional amino acid substitutionat position 77, 92, or 109 (“AA change”), generated as describedpreviously. These variants were analyzed for increased light output asdescribed previously, i.e., screened for variants that were at least1.3× brighter than C1+A4E, using native coelenterazine, knowncoelenterazine-hh, and novel coelenterazines PBI-3939, PBI-3894,PBI-3896, PBI-3897, PBI-3932, or PBI-3925 as a substrate. The followingadditional substitutions yielded a variant that was at least 1.3×brighter than C1+A4E: L92G, L92Q, L92S, L92A, L92M, L92H, L92Y, F77W,F77Y, F77S, F77T, F77V, F77A, F77G, F77C, F77D, F77M, and Y109F. Asshown in Table 5, L92H, F77W and F77A substitutions had the mostdramatic improvements with PBI-3897, PBI-3896, and PBI-3932.

TABLE 5 Site Saturation of Positions 77, 92 and 109 AA PBI- PBI- PBI-PBI- PBI- Change native hh PBI-3939 3894 3896 3897 3932 3925 L92G 2.2L92Q 2 1.8 1.6 1.3 1.4 2.8 1.4 3.4 L92S 2.9 1.5 2.9 2.7 6 L92A 2.5 1.3L92M 1.3 L92H 2.2 21 9.1 3.4 5.9 L92Y 2.5 F77W 1.4 1.4 1.4 8.3 3.2 1.72.3 F77Y 1.6 1.3 4.9 6.5 F77S 2.6 F77T 2.3 F77V 2.3 F77A 7.9 2.5 F77G3.1 F77C 2.3 F77D 1.5 F77M 1.5 1.6 Y109F 1.34 14

Additional C1+A4E variants (Group A) were generated by site-directedmutagenesis as described previously to have an additional substitutionin at least one of the following amino acid positions relative to SEQ IDNO: 1: 18, 20, 54, 59, 72, 77, 89, 92, 109, 113, 127, 136, or 164. Theseamino acid positions were chosen because, based on the primary andsecondary screens of Library 1, substitutions at these positions hadincreased total light output compared to C1+A4E using at least one ofthe following as a substrate: novel coelenterazines PBI-3841, PBI-3896,PBI-3897, PBI-3894, PBI-3925, or PBI-3932, or known coelenterazines2-methyl coelenterazine or PBI-3912. FIG. 7 lists the variants (“Clone”)and the additional amino acid substitutions contained in each variant.Variant clones were assayed in triplicate as described for the secondarymanual screen as described previously and normalized to C1+A4E. FIGS.8A-B and 9 show the normalized average luminescence of the variantslisted in FIG. 7 with various coelenterazines as substrates. FIGS. 8A-Band 9 show variants with either large increases in luminescence for thelisted novel compounds compared to C1+A4E or no change or a decrease inluminescence for the known coelenterazine compared to C1+A4E. CloneQC27, which has additional amino acid substitutions Q18L, F54I, L92H,and Y109F, had a 561.32-fold-increase in luminescence with PBI-3896, a392.98-fold-increase with PBI-3894, and a 283.85-fold-increase withPBI-3896 compared to C1+A4E. This data shows that Q18L, L92H, and Y109Fcan be combined with each other and with additional substitutions toresult in variants with improved relative specificity.

Other substitutions of interest identified from Library 1 were combinedto generate additional variants (Group B) (FIG. 10). Additional aminoacid substitutions were made in at least one of the following amino acidpositions relative to SEQ ID NO: 1: 18, 20, 54, 71, 77, 90, 92, 109, or127. These substitutions showed improvement with at least one of thefollowing novel coelenterazines as a substrate: PBI-3841, PBI-3896,PBI-3897, PBI-3894, PBI-3925, or PBI-3932. These variants were assayedas described for Group A variants using native coelenterazine, knowncoelenterazine-hh, and novel coelenterazines PBI-3939, PBI-3945,PBI-3840, PBI-3932, PBI-3925, PBI-3894, and PBI-3896. Variant cloneswere assayed in triplicate as described for the secondary manual screenas described previously and normalized to C1+A4E. FIG. 11 shows thenormalized average luminescence of the variants listed in FIG. 10 withthe various coelenterazines as substrates. FIG. 11 shows variants witheither large increases in luminescence for the listed novelcoelenterazines compared to C1+A4E or no change or a decrease inluminescence for the native and known coelenterazine compared to C1+A4E.

Additional variants were generated with the additional amino acidsubstitution 190V and/or Y109F (Group C) and compared to variantsgenerated from Group A or B (see FIG. 12). Clones containing variantswith an 190V substitution (190V″), a Y109F substitution (“Y109F”), orboth substitutions (“LE2”) were compared to clones QC #27, QC#2 E7, QC#2F4, and QC#1 A11 using assays as described for Group A recombinants withnative coelenterazine, known coelenterazine-hh, and novelcoelenterazines PBI-3939, PBI-3945, PBI-3889, PBI-3840, PBI-3925,PBI-3932, PBI-3894, PBI-3896, and PBI-3897 as substrates (FIG. 12).Variant clones were assayed in triplicate as described for the secondarymanual screen as previously described and normalized to C1+A4E (FIG.12). FIG. 12 shows variants with either large increases in luminescencefor the listed novel coelenterazines compared to C1+A4E and no change ora decrease in luminescence for the native or known coelenterazinecompared to C1+A4E. FIG. 12 shows that 190V provided greater lightoutput for native coelenterazine and several of the novel substrates.

B. QC27 Variants

The variant QC27 (SEQ ID NOs: 4 and 5) from A, which has additionalamino acid substitutions Q18L, F54I, L92H, and Y109F, was cloned into apF4A modified vector as described previously to create a C-terminal HT7(Promega Corp.) fusion protein (“QC27-HT7”) (SEQ ID NOs: 44 and 45).4400 variants of QC27-HT7 (Library 2) were generated by randommutagenesis as described previously, and primarily screened forincreased relative specificity change as described previously usingnative coelenterazine and novel coelenterazines PBI-3896 and PBI-3897 assubstrates. Variant clones were selected, sequenced, and assayed in asecondary manual screen as described previously using nativecoelenterazine, known coelenterazine-hh, and novel coelenterazinesPBI-3897, PBI-3896, and PBI-3894 as substrates.

FIG. 13 lists the additional amino acid substitutions (“Sequence”)identified in these variants (“Sample”), and the luminescence of thevariants using native coelenterazine, known coelenterazine-hh, and novelcoelenterazines PBI-3897, PBI-3896, and PBI-3894 as substrates in thesecondary screen normalized to the corresponding starting QC27-HT7. Thevariants in FIG. 14, had at least one of the following additional aminoacid substitutions: F1I, R11Q, L18I, L18Q, V21L, V21M, L22F, F31I, Q32H,V45E, L46Q, S47P, G48R, E49D, G51E, D55E, G67S, F68Y, F68L, Q69H, L72Q,E74K, E74I, M75K, I76F, I76V, H86R, I90T, H92Q, H92R, T96A, V98F, I99V,I99T, V102M, M106I, F109Y, L142V, V158I, T159S, L168F, or G170R (theG170R is located in the linker region between HT7 and the OgLucvariant).

The amino acid substitutions F68Y in variant 24B12, L72Q in variant29C4, and M75K in variant 3H11 each provided greater light output fornative coelenterazine and several of the novel substrates. The aminoacid substitutions V21L in variant 25A11 and H92R in variant 1B6provided improved relative specificity. Both of these substitutions werecases where luminescence signals were down using the novelcoelenterazines as substrates, but were down more using native and knowncoelenterazines as substrates.

Additional QC27-HT7 variants were generated to have specific amino acidsubstitutions (FIG. 14) using site-directed mutagenesis as describedpreviously. Additional substitutions were made in at least one of thefollowing amino acid positions relative to SEQ ID NO: 1: 21, 68, 72, 75,76, 90, 92, and 158, as these positions showed improvement in relativespecificity change as shown in FIG. 14. FIG. 15 shows the luminescenceof the QC27-HT7 variants using native coelenterazine, knowncoelenterazine-hh, and novel coelenterazines PBI-3897, PBI-3841,PBI-3896, and PBI-3894 as substrates normalized to the correspondingstarting QC27-HT7. As seen in FIG. 15, combining the three amino acidsubstitutions F68Y, L72Q, and M75K with V1581, as for example in variantQC27#1, provided greater light output for each coelenterazine tested.

C. QC27-9a Variants

The variant QC27-9a (SEQ ID NOs: 6 and 7) from B, a QC27-HT7 fusionprotein with additional amino acid substitutions V21L, H29R, F68Y, L72Q,M75K, and V158I, was used as a starting sequence to generate a library.4400 variants of QC27-9a (Library 3) were generated by randommutagenesis as described previously and screened for increased relativespecificity change using native coelenterazine and novel coelenterazinesPBI-3841 and PBI-3897. Variant clones were selected, sequenced, andassayed in a secondary manual screen as described previously usingnative coelenterazine, known coelenterazine-hh, known coelenterazine-h,and novel coelenterazines PBI-3841 and PBI-3897 as substrates. FIG. 16lists the additional substitutions (“AA change”) identified in thevariants (“Sample”), and the average luminescence of the variants usingnative coelenterazine, known coelenterazine-hh, known coelenterazine-h,and novel coelenterazines PBI-3841 and PBI-3897 as substrates in thesecondary screen normalized to the corresponding starting QC27-9a. Theincrease in relative specificity represents cases where there was adecrease in luminescence for the variant with the novel, native, andknown coelenterazines compared to the starting template, butluminescence with the native and known coelenterazines decreased more.For example, the variant 30D12 with the amino acid substitution L22F hadan approximately three-fold loss in activity with the novelcoelenterazines PBI-3841 and PBI-3897. However, with nativecoelenterazine, known coelenterazine-h, and known coelenterazine-hh, theluminescence of the variant 30D12 was down by ten-fold or more.

FIG. 17 shows a comparison of the luminescence of C1+A4E, QC27-HT7 andQC27-9a compared to humanized Renilla luciferase (referred herein as“hRL”) (SEQ ID NOs: 30 and 31) using native coelenterazine, knowncoelenterazine-hh, and novel coelenterazines PBI-3841 and PBI-3897 assubstrates. Although the reaction of QC27-9a with PBI-3897 was brighterthan QC27-9a with PBI-3841 (see FIG. 17), the evolution trend, i.e.,magnitude of improvement in luminescence, was greatest for PBI-3841(Table 6). Combining the improvement in luminescence (440-fold) with thedecrease in luminescence for native coelenterazine (800-fold) indicateda change in relative specificity (350,000-fold) of QC27-9a usingPBI-3841 compared to native coelenterazine.

TABLE 6 The Change in Relative Specificity of the OgLuc Variants forPBI-3897 and PBI-3841 Compared to Native Coelenterazine andcoelenterazine-hh. Evolution trend: Change in relative specificity C1A4Eto (novel coelenterazine/ Compound QC27 #9a native coelenterazine)coelenterazine DOWN 800X coelenterazine-hh DOWN 300X PBI-3897 UP 100X 80,000X PBI-3841 UP 440X 350,000X

D. IVY Variants

IVY (SEQ ID NOs: 8 and 9), a C1+A4E variant with additional amino acidsubstitutions F54I, I90V, and F77Y, was cloned into a pF4A modifiedvector as described previously to create a C-terminal HT7 fusion protein(“IVY-HT7”). 4400 variants of IVY-HT7 (Library 4) were generated byrandom mutagenesis and screened for increased light output (i.e.,increased brightness) and increased relative specificity using nativecoelenterazine, known coelenterazine-hh, and novel coelenterazinesPBI-3840, PBI-3889, PBI-3925, PBI-3932, and PBI-3945 as substrates.Variant clones were selected, sequenced, and assayed in triplicate in asecondary screen as described previously using native coelenterazine,known coelenterazine-hh, and novel coelenterazines PBI-3889, PBI-3939,PBI-3945, and PBI-4002 as substrates. FIGS. 18 and 19 lists theadditional substitutions (“AA change”) identified in the variants(“Sample”) and the average luminescence of the variants normalized toIVY-HT7 using native coelenterazine, known coelenterazine-hh, and novelcoelenterazines PBI-3889, PBI-3939, PBI-3945, and PBI-4002 as substratesin the secondary screen. FIG. 18 lists those variants chosen based onperformance with PBI-3945 (Group A), which had at least one of thefollowing amino acid substitutions: Q18H, D19N, Q20P, Q32P, K33N, V38I,V38F, K43N, I44F, E49G, I60y, Q69H, I76N, Y77N, Y94F, G95S, G95D, F110I,V119M, K124M, L149I, or R152S. FIG. 19 lists those variants chosen basedon performance with PBI-3889 (Group B), which had at least one of thefollowing amino acid substitutions: F6Y, Q18L, L27V, S28Y, Q32L, K33N,V36E, P40T, Q42H, N50K, G51R, H86L, N135D, or I155T.

Additional IVY-HT7 variants were generated to have additional specificamino acid substitutions using site-directed mutagenesis as describedpreviously. FIG. 20 lists variants with at least one of the followingadditional amino acid positions relative to SEQ ID NO: 1: 19, 20, 27,32, 38, 43, 49, 58, 77, 95, 110, and 149, as these substitutions wereidentified in the variants of FIG. 18, which showed specificity towardsPBI-3945 and PBI-4002. FIG. 21 shows the luminescence of the variantslisted in FIG. 20 normalized to IVY-HT7 using native coelenterazine,known coelenterazine-h, known coelenterazine-hh, and novelcoelenterazines PBI-3939, PBI-3945, PBI-4002, PBI-3932 and PBI-3840 assubstrates. None of the variants showed an improvement over IVY-HT7, butthere were instances, such as variant C5.19 (SEQ ID NOs: 12 and 13)where luminescence with native or a known coelenterazine decreased about3-4 logs, but luminescence with PBI-3945 and PBI-4002 decreased onlytwo-fold. Variant C5.19 has additional amino acid substitutions L27V,V38I, and L149I.

FIG. 22 lists variants with at least one of the following additionalamino acid positions relative to SEQ ID NO: 1: 6, 18, 27, 28, 33, 34,36, 40, 50, 51, 135, and 155, as these substitutions were identified inthe variants of FIG. 19, which showed specificity towards PBI-3889 andPBI-3939. FIG. 23 shows the luminescence of the variants listed in FIG.21 using native coelenterazine, known coelenterazine-h, knowncoelenterazine-hh, and novel coelenterazines PBI-3939, PBI-3945,PBI-3889, PBI-4002, PBI-3932, and PBI-3840 as substrates normalized toIVY-HT7. Luminescence decreased for each of the variants compared toIVY-HT7. Variant C1.3 (SEQ ID NOs: 10 and 11) had about 2000-fold moreluminescence with PBI-3939 than with native or known coelenterazine.Variant C1.3 has additional amino acid substitutions F6Y, K33N,N135D,and 1155T.

The best IVY-HT7 variants for relative specificity change compared tohRL and IVY-HT7 were C5.19, which had the best luminescence withPBI-3945, and C1.3, which had the best luminescence with PBI-3889. FIG.24 shows the luminescence of hRL, IVY-HT7, C5.19 (a C-terminal HT7fusion), and C1.3 (a C-terminal HT7 fusion) with native coelenterazine,known coelenterazine-h, known coelenterazine-hh, and novelcoelenterazines PBI-3939 and PBI-3945.

E. IV Variants

IV (SEQ ID NOs: 14 and 15), a C1+A4E variant with additional amino acidsubstitutions F54I and I90V, was generated as previously described. Todetermine the brightest variant for use as a transcriptional reporter,luminescence was measured as described previously provided for C1+A4E(SEQ ID NOs: 2 and 3), IVY (SEQ ID NOs: 8 and 9), and IV (SEQ ID NOs: 14and 15) using native coelenterazine, known coelenterazine-hh, and novelcoelenterazines PBI-3939, PBI-3945, PBI-3889, and PBI-4002 assubstrates. hRL was used as a control. As seen in FIG. 25, IV wasbrighter than both C1+A4E and IVY. The amino acid substitution F54I inIV provided greater light output for native coelenterazine and severalof the novel substrates. All three variants were brighter than hRL withthe tested coelenterazines.

The data from A, B and D (i.e., screenings of the libraries generatedfrom C1+A4E, IVY, and QC27 as the starting sequences) were reviewed todetermine those additional amino acid substitutions with increased lightoutput (i.e., increased brightness) with a variety of coelenterazines.IV variants were generated as described previously to have additionalsubstitutions which had reduced specificity for native coelenterazine bytwo- to ten-fold. As listed in FIG. 26, the IV variants (“clone”) had anadditional amino acid substitution (“Sequence”) of at least one of thefollowing amino acid substitutions: F1I, E4K, Q18L, L27V, K33N, V38I,F68Y, M70V, L72Q, M75K, or V102E.

Sixteen plates of variant clones for all combinations of amino acidsubstitutions were primarily screened and assayed using the automatedrobot method described previously with native coelenterazine, knowncoelenterazine-h, known coelenterazine-hh, and novel coelenterazinesPBI-3889 and PBI-3945 as substrates. Variants with improved luminescencewere selected, sequenced, and assayed in triplicate using the manualscreen as described previously. Luminescence was measured using nativecoelenterazine, known coelenterazine-h, known coelenterazine-hh, andnovel coelenterazines PBI-3889, PBI-3939, PBI-3945, and PBI-4002 assubstrates. Corresponding starting sequences IV and hRL were used ascontrols.

FIG. 26 lists the variants, and the additional amino acid substitutionsidentified in the variants. FIG. 27 shows the average luminescence ofthe variants in the secondary screen normalized to IV. Variant 8A3 (SEQID NOs: 26 and 27), which has additional amino acid substitutions F1I,L27V, and V38I, had improved relative specificity with novelcoelenterazines, but was not brighter than IV. Variant 8F2 (SEQ ID NOs:46 and 47), which has additional amino acid substitution L27V, offeredimproved relative specificity and brightness with 3 of the 4 novelcoelenterazines used. Variant 9B8 (SEQ ID NOs: 18 and 19), which hasadditional amino acid substitutions Q18L, F68Y, L72Q, and M75K, wasbrighter for all substrates and offered some relative specificityadvantage over native coelenterazine as well. Variant 9F6 (SEQ ID NOs:20 and 21), which has additional amino acid substitutions Q18L, L27V,V38I, F68Y, L72Q, and M75K, showed similar improvements as was seen with8F2. Variant 15C1 (SEQ ID NOs: 16 and 17), which has additional aminoacid substitutions E4K, K33N, F68Y, L72Q, and M75K, was brighter for allnovel coelenterazines, but did not have any improved relativespecificity benefit. The amino acid substitution Q18L in variant 1D6provided improved relative specificity, i.e., away from nativecoelenterazine and towards novel substrates, in the context of IV. Ingeneral, the amino acid substitution L27V provided improved relativespecificity in the context of IV.

FIG. 28 shows the luminescence of the 8A3, 9B8, 9F6, and 15C1 variantsin the secondary screen using native coelenterazine, knowncoelenterazine-hh, known coelenterazine-h, and novel coelenterazinesPBI-3939, PBI-3945, PBI-3889, and PBI-4002 as substrates compared to IVand hRL. Variant 8A3 had 2 logs decrease in brightness with nativecoelenterazine compared to IV. Variant 9F6 had 1 log decrease inbrightness with native coelenterazine compared to IV. Variant 15C1 withPBI-3945 was the brightest, but the signal half-life was short (seeExample 27).

F. 9B8 Variants

The 9B8 variant from E was further modified to generate additionalvariants with increased light emission and/or improved relativespecificity for PBI-3939. Amino acid substitution L72Q appeared to be abeneficial amino acid substitution for increased light emission (i.e.,brightness) as this substitution was identified in the variants 9B8,9F6, and 15C1, all of which showed improved light emission. To determineif other amino acid substitutions at position 72 would provide similarincreases in brightness, additional variants of 9B8 were generated asdescribed previously by saturating position 72 with alternativeresidues. Four replicates of E. coli lysates were prepared and analyzedfor brightness as described previously using PBI-3939 as a substrateexcept the assay buffer contained 10 mM CDTA, 150 mM KCl, 10 mM DTT, 100mM HEPES, pH 7.0, 35 mM thiourea, and 0.5% TERGITOL® NP-9 (v/v). Table 7lists 9B8 variants (“Variant”) with similar or improved luminescencecompared to 9B8 as indicated by luminescence normalized to 9B8 (“RLU(normalized to 9B8)”), i.e., fold improvement. The amino acidsubstitutions of A, G, N, R, and M at position 72 provided at least thesame brightness benefit as amino acid Q, i.e., 1-fold.

TABLE 7 Variants with Similar Luminescence Compared to Variant 9B8.Variant RLU (normalized to 9B8) 9B8 + Q72A 1.1 9B8 + Q72G 1 9B8 + Q72N 19B8 + Q72R 1 9B8 + Q72M 1

Additional variants with improved relative specificity to novel PBI-3939were generated as described previously by saturating amino acidpositions 18, 68, 72, 75, and 90 in variant 9B8. E. coli lysates wereprepared and analyzed for brightness as described previously usingnative coelenterazine and novel PBI-3939 as substrates. Relativespecificity was determined from the ratio of the luminescence of thevariant with PBI-3939 to the luminescence of the variant with nativecoelenterazine, normalized to the ratio of corresponding luminescence of9B8. Table 8 lists 9B8 variants (“Variant”) with at least 1.1×fold-increase in relative specificity for PBI-3939. The resultsdemonstrate that at least one additional change at each of the sitesprovided improved relative specificity for PBI-3939 versus nativecoelenterazine. 9B8 variants with amino acid substitutions K, D, F, G,Y, W, and H at position 18 had the highest fold improvement in relativespecificity.

TABLE 8 Variants with Improved Relative Specificity for PBI-3939Relative specificity (PBI-3939 RLU/native coelenterazine RLU; Variantnormalized to 9B8) 9B8 + L18K 40.7 9B8 + L18D 25.8 9B8 + L18F 25.6 9B8 +L18G 18.2 9B8 + L18Y 17.8 9B8 + L18W 11.2 9B8 + L18H 9.1 9B8 + L18R 3.59B8 + L18M 3.4 9B8 + L18N 2.9 9B8 + L18P 2.6 9B8 + L18S 2.3 9B8 + Y68W1.1 9B8 + Q72W 6.1 9B8 + Q72Y 2.5 9B8 + Q72F 2.2 9B8 + Q72V 2.2 9B8 +Q72I 2.1 9B8 + Q72T 1.9 9B8 + Q72N 1.8 9B8 + Q72R 1.7 9B8 + Q72P 1.69B8 + Q72G 1.5 9B8 + Q72A 1.4 9B8 + Q72M 1.3 9B8 + Q72C 1.3 9B8 + Q72H1.2 9B8 + Q72S 1.2 9B8 + M75F 1.2 9B8 + V90R 2.4 9B8 + V90Y 1.6 9B8 +V90D 1.4 9B8 + V90P 1.4 9B8 + V90K 1.3 9B8 + V90Q 1.2

G. 9B8+K33N Variants

An additional variant, 9B8 opt+K33N (SEQ ID NOs: 42 and 43) wasgenerated to investigate the benefits of amino acid substitution K33Nfor brightness, relative specificity, and thermal stability. 9B8opt+K33N was examined and compared to 9B8 opt (described in Example 25A)in various applications.

E. coli lysates containing the variant 9B8 opt or 9B8 opt+K33N wereprepared and analyzed as described previously except the assay buffercontained 0.1% TERGITOL® NP-9 (v/v). Luminescence generated from thelysates was measured using the novel PBI-3939 and native coelenterazineas substrates. The relative specificity of the variants for PBI-3939 andnative coelenterazine was calculated as described previously. 9B8opt+K33N (“K33N”) had greater light output (RLU) and a higher relativespecificity for PBI-3939 than native coelenterazine compared to 9B8 opt(FIG. 29), indicating that the K33N substitution provided greater lightoutput and improved relative specificity.

A new OgLuc library was created using 9B8opt+K33N as a startingtemplate. The random library was created using DIVERSIFY® PCR RandomMutagenesis Kit (ClonTech; Catalog #630703). Condition 5 (as listed inthe user manual) was used to generate additional variants, and theaverage mutation rate was calculated to be 2.6 mutations per gene bycompiling sequence data from 83 randomly selected clones. This PCRlibrary was cloned into the pF4Ag-based, non-fusion vector backgrounddescribed previously and the sandwich background, i.e., Id-OgLuc-HT7(described in Example 45). Variants in the pF4Ag-base non-fusion vectorbackground are designated with (NF). Variants in the sandwich vectorbackground are designated with (F). In order to clone the PCR productinto both vectors, an amino acid, i.e., a glycine, was appended to thevariant sequence in pF4Ag, generating a new position 170 in the OgLucvariant (“170G”). The 170G is present in the sandwich construct, but inthis case is considered part of the linker between OgLuc and HT7. Foreach library, 4,400 E. coli clones were assayed as described previouslywith the following exceptions. The lysis buffer contained 300 mM MES pH6.0 instead of HEPES, and 0.5% TERGITOL® NP-9 (v/v), but did not containthiourea. The assay buffer contained 100 mM MES pH 6.0 instead of HEPES,and 35 mM thiourea. The assay volumes were as follows: 10 μL cells, 40μL lysis buffer, and 50 μL assay buffer.

The PCR library in the pF4Ag-based non-fusion background was screenedfor additional variants with increased luminescence compared to 9B8opt+K33N+170G (SEQ ID NOs: 68 and 69). Selected variants were thenassayed in HEK293 and NIH3T3 cells. For each cell type, 15,000 cellswere plated and grown overnight at 37° C. The next day, the cells weretransfected as described in Example 25 with 10 ng pGL4.13 (PromegaCorp.) as a transfection control and 100 ng of the OgLuc test DNA. Themedia was removed, and the cells were lysed with 100 μL of lysis bufferas described in Example 25 except the lysis buffer contained 100 mM MESpH 6.0 instead of HEPES, and luminescence measured using a GLOMAX®Luminometer. For each sample, 10 μL of lysate was assayed with 50 μl oflysis buffer containing 20 μM PBI-3939. For the transfection control, 10μL of lysate was assayed with 50 μL of BRIGHT-GLO™ Assay Reagent.

Table 9 shows the fold-increase in luminescence of variants in E. coli,HEK293, and NIH3T3 cells and the amino acid substitutions found in thevariants. The variants 27A5 (NF) (SEQ ID NOs: 70 and 71), 23D4 (NF) (SEQID NOs: 72 and 73) and 24C2 (NF) (SEQ ID NOs: 74 and 75) had at least1.3 fold-increase in luminescence in E. coli and HEK293 cells.

TABLE 9 Increase in Luminescence Generated by OgLuc Variants Compared to9B8 opt + K33N + 170G in E. coli, HEK293 and NIH3T3 Cells Fold over 9B8opt + K33N + 170G Sample Sequence E. coli HEK293 NIH3T3 27A5 (NF) T39T,170G 1.3 1.5 1.3 23D4 (NF) G26G, M106L, R112R, 170G 1.5 1.6 1.2 24C2(NF) R11Q, T39T, 170G 1.5 1.5 1.1

Based on the above data, further combination variants were designed andgenerated (see Table 10) in the context of the pF4Ag-based non-fusionvector background without the 170G. The variants were analyzed in E.coli, HEK293 and NIH3T3 cells as described above and compared to 9B8opt+K33N. The variants were also examined for luminescence with nativecoelenterazine. Table 10 shows the fold-increase in luminescence of thevariants in E. coli, HEK293, and NIH3T3 cells, and the amino acidsubstitutions found in the variants (“Sample”). The variants were namedby adding the additional amino acid substitutions in the variant to theprefix “9B8 opt+K33N.” Table 11 shows the relative specificity of thedifferent variants for PBI-3939 compared to native coelenterazine in E.coli, NIH3T3, and HEK293 cells. As shown in Table 10, the variant 9B8opt+K33N+T39T+K43R+Y68D (“V2”; SEQ ID NOs: 92 and 93) had improvedluminescence in E. coli and a slight improvement in luminescence inNIH3T3 cells. The variant 9B8 opt+K33N+L27V+K43R+Y68D (“L27V, K43R,Y68D”) had a neutral improvement in luminescence (Table 10) and 5×fold-increase in relative specificity over 9B8 opt+K33N (Table 11) inthe three cell types examined.

TABLE 10 Increase in Luminescence Generated by OgLuc CombinationVariantsCompared to 9B8 opt + K33N in E. coli, NIH3T3 and HEK293 Cells Fold over9B8 opt + K33N Sample E. coli NIH3T3 HEK293 T39T 1.8 1.1 1.1 K43R 1.21.1 1.1 T39T, K43R 1.3 0.9 1.1 Y68D 1.0 1.0 1.2 K43R, Y68D 1.2 1.2 1.2T39T, K43R, Y68D (“V2”) 1.8 1.1 1.3 L27V 0.9 0.7 0.8 L27V, K43R 0.7 0.60.6 L27V, K43R, Y68D 1.2 0.8 0.9 L27V, Y68D 1.2 0.8 0.7 S66N, K43R 0.91.1 1.1 L27V, K43R, S66N 1.0 0.6 0.7 9B8 opt + K33N 1.0 1.0 1.0

TABLE 11 Change in Relative Specificity of OgLuc Combination Variantsfor PBI-3939 Compared to Native Coelenterazine in E. coli, NIH3T3 andHEK293 Cells Fold over Native Coelenterazine Sample E. coli NIH3T3HEK293 T39T 18.2 18 20 K43R 29.5 31 32 T39T, K43R 29.4 30 32 Y68D 11.410 12 K43R, Y68D 18.6 19 21 T39T, K43R, Y68D (“V2”) 18.5 18 21 L27V 85.285 97 L27V, K43R 120.1 131 147 L27V, K43R, Y68D 98.3 98 101 L27V, Y68D59.9 61 64 S66N, K43R 22.9 23 25 L27V, K43R, S66N 100.4 97 106 9B8 opt +K33N 19.0 19 19

Additional OgLuc variants were generated from 9B8 opt+K33N to contain atleast one of the following additional amino acid substitutions relativeto SEQ ID NO: 1: L27V, T39T, K43R, Y68D, or S66N (see “Sample” in Table12 for the amino acid substitutions in the variants). The variants werenamed by adding the additional amino acid substitutions in the variantafter the prefix “9B8 opt+K33N.” These additional variants and thevariants 9B8 opt+K33N+L27V+Y68D (“L27V, Y68D”), 9B8opt+K33N+L27V+K43R+Y68D (“L27V, K43R, Y68D”), 9B8opt+K33N+L27V+K43R+S66N (“L27V, K43R, S66N”), and 9B8opt+K33N+T39T+K43R+Y68D (“T39T, K43R, Y68D”; also known as “V2”) fromabove, were examined for brightness, relative specificity, signalstability and thermal stability. The variants were compared to thevariants 9B8 opt (“9B8”) and 9B8 opt+K33N (“K33N”).

E. coli lysates containing the variants were prepared and analyzed asdescribed previously. Luminescence generated from the lysates wasmeasured using the novel PBI-3939 and native coelenterazine assubstrates. The luminescence of the variants was normalized to theluminescence generated by 9B8 opt (Table 12). The relative specificityof the variants for PBI-3939 and native coelenterazine was calculated bydividing the luminescence of the variants using PBI-3939 as a substratewith the luminescence of the variants using native coelenterazine as asubstrate (Table 12). This data indicates that the amino acidsubstitution L27V lowers specificity for native coelenterazine.

TABLE 12 Increase in Luminescence Generated by OgLuc Variants Comparedto 9B8 and Change in Specificity of OgLuc Variants for PBI-3939 Comparedto Native Coelenterazine in Bacterial Lysates Sample Fold over 9B8 Foldover coelenterazine 9B8 1.0 7 K33N 1.1 21 T39T, Y68D 0.9 12 T39T, L27V,K43R 1.2 149 L27V, T39T, K43R, Y68D 1.8 110 T39T, K43R, Y68D 1.6 21L27V, T39T, K43R, S66N 1.3 114 L27V, K43R, Y68D 1.3 110 L27V, Y68D 1.063 L27V, K43R, S66N 1.1 114

H. V2 Variants

A set of additional variants were designed using V2 (9B8opt with theadditional amino acid substitutions K33N+T39T+K43R+Y68D) as a template.The substitutions shown in Table 13 were designed based on either 1) theknown diversity according to the structure-based alignment of 28 fattyacid binding proteins (1VYF, 1FDQ, 2A0A, 1O8V, 1BWY, 2ANS, 1VIV, 1PMP,1FTP, 2HNX, 1JJJ, 1CBR, 2CBS, 1LPJ, 1KQW, 2RCQ, 1EII, 1CRB, 1IFC, 2PYI,2JU3, 1MVG, 2QO4, 1P6P, 2FT9, 1MDC, 1O1U, 1EIO; See U.S. PublishedApplication No. 2010/0281552), or 2) the probing of alternative residuesat positions previously identified to play a role in substratespecificity. Changes listed under “Consensus” in Table 13 relate toresidues identified in at least 50% of the aligned, above-mentionedfatty acid binding proteins. Changes listed under “Predominant Minority”relate to residues identified in many of the fatty acid binding proteinsmentioned above, but found in fewer than 50% of the aligned sequences.Changes listed under “Other” relate to residues were identified lessfrequently than the predominant minority residue at a given position inthe aligned sequences. Finally, changes listed under “Specificity”relate to positions suspected to be involved in determining a variant'sspecificity for coelenterazine or a coelenterazine analog. For example,the designed specificity changes at position 27 (leucine residue in theparental sequence, i.e., V2) were changed to other hydrophobic residuesor amino acids representing alternative chemistries (e.g., otherhydrophobic residues containing rings, residues containing unchargedpolar side chains, or residues containing charged side chains); and thedesigned specificity changes at position 40 (proline in the parentalsequence), were to a sampling of different chemistries (chemistries(e.g., other hydrophobic residues containing rings, residues containinguncharged polar side chains, or residues containing charged sidechains); note that glycine, glutamine, isoleucine, and leucine areidentified at this position the aligned fatty acid binding proteins).

TABLE 13 Predominant Consensus minority Other Specificity  9 T  9 K  9 R27 A, I, M, G, D  14 S  10 Y 40 G 40 T, S, F, D, Y  16 E  23 R  22 M  32I  23 K  63 T  24 A  87 T  25 L 100 I  32 Et 111 N, D  35 A 118 I  39 K134 K  46 Q 142 K, W  57 F 147 N  63 S 149 M  87 N 152 E  97 E 162 Q  98F 100 E 102 T 110 D 113 K 118 V 125 L 126 V 129 Q 130 K 142 E 146 G 147D 150 V 152 T 165 K

The variants were constructed using standard site-directed mutagenesisprotocols (see previous examples), and the resulting plasmidstransformed into E. coli for analysis. Cultures were grown per standardwalk away induction in minimal media as described previously. To 10 μLof the cultured, transformed E. coli cells, 40 μL of lysis buffer (100mM MES pH 6.0, 0.3×PLB, 0.3 mg/mL Lysozyme, 0.003 U/μL RQ1 DNaseI and0.25% TERGITOL® NP-9 (v/v)) was added followed by the additional of anequal volume (50 μL) of assay reagent (1 mM CDTA, 150 mM KCl, 2 mM DTT,20 μM PBI-3939 or native coelenterazine, 100 mM MES pH 6.0, 35 mMthiourea, and 0.5% TERGITOL® NP-9 (v/v)). Luminescence was measured on aGLOMAX® 96 Microplate Luminometer (Promega Corp.).

Table 14 summarizes the different amino acid substitutions identified inthe analysis. The data is presented as normalized to the parental clone(V2) with regards to the luminescence measured for both PBI-3939 andnative coelenterazine. The relative change in specificity to PBI-3939with respect to native coelenterazine is also shown.

TABLE 14 RELATIVE CHANGE IN PBI-3939 COELENTERAZINE SPECIFICITYSubstitution NORMALIZED NORMALIZED FOR PBI-3939  10 Y 0.7 0.2 3.5  14 S1.3 1.2 1.1  16 E 0.5 0.2 2.5  23 K 1.3 4 0.3  24 A 0.4 0.2 2.0  25 L0.0001 0.000023 4.3  27 A 0.8 0.1 8.0  27 D 0.006 0.001 6.0  27 G 0.0880.005 17.6  27 I 0.2 0.024 8.3  27 M 2.2 0.9 2.4  40 I 0.0017 0.0002 8.5 40 L 0.0007 0.0001 7.0  40 Q 0.0001 0.000026 3.8  87 N 1.2 1.5 0.8  87T 1.3 1.6 0.0  97 E 0.014 0.01 1.4 100 I 0.002 0.002 1.0 102 T 1.1 1.11.0 111 N 0.6 0.6 1.0 113 K 1.2 0.6 2.0 125 L 0.6 0.4 1.5 129 Q 0.00030.0001 3.0 130 K 1.1 0.9 1.2 142 E 0.9 0.3 3.0 142 K 0.9 0.3 3.0 142 W0.8 0.4 2.0 146 G 0.9 0.8 1.1 147 N 0.4 0.4 1.0 149 H 0.7 0.4 1.8 150 V0.9 0.4 2.3 152 E 0.9 0.5 1.8 152 T 0.9 0.3 3.0

I. L27V Variants

Using the OgLuc variant L27V as a starting template, i.e., startingsequence or parental sequence, additional variants were made in whichsome of the amino acids (Table 15) in the L27V variant were reverted tothe amino acids found in the native OgLuc luciferase of SEQ ID NO: 1.The variants were constructed by site-directed mutagenesis as previouslydescribed. The variants were then screened as previously described forrelative activity with either native coelenterazine or PBI-3939.Luminescence was measured on a TECAN® INFINITE® F500 5 min aftersubstrate/assay reagent (as described in H) was added and normalized tothe L27V variant starting template. SDS-PAGE analysis of the lysatesindicates comparable expression levels (data not shown).

Table 15 shows the relative activities of the L27V variants with nativecoelenterazine or PBI-3939. Relative activities <1 indicate thereversion is detrimental compared to the residue at that site in theL27V variant. Relative activities >1 indicate the reversion is favorablefor activity compared to the residue at that site in the L27V variant.Some additional data on these mutants indicated the following: 166K,54F, 54A and L27V were tested for thermal stability. The T_(1/2) 60° C.for 166K, 54F, and 54A were 87, 74, and 33%, respectively, indicatingthese substitutions cause a reduction in thermal stability. The Kmvalues for these same 4 variants were the following: for nativecoelenterazine, L27V was 16 μM, 54A was 23 μM, 54F was 40 μM, and 166Kwas 21 μM; for PBI-3939, L27V was 18 μM, 54A was 62 μM, 54F was 163 μM,and 166K was 23 μM. This indicates higher substrate affinity for L27V,particularly for the position 54 substitutions.

TABLE 15 Native coelenterazine (50 mM) PBI-3939 (50 mM) AA Relativeactivity AA Relative activity substitution (5 min) substitution (5 min) 72L 0.2 72L 0.6  4A 1.0 4A 1.0 124Q 1.6 124Q 1.0  43K 1.9 43K 1.1 115P0.6 115P 0.9 166N 2 166N 2.0  75M 1.1 75M 1.2  54F 0.1 54F 0.4  68F 0.568F 0.9  33A 1.7 33A 1.0 138Y 1.0 138Y 1.0  54A 0.6 54A 1.6  90I 0.8 9010.6  33K 4.2 33K 0.8  44V 0.7 44V 1 166K 2.1 166K 2  11Q 1.6 11Q 1.3166F 0.3 166F 0.4  18Q 4.1 18Q 0.6

Example 23 Mutational Analysis of Position 166

A. To assess the effect of different amino acids at position 166 on theluciferase activity, the arginine (R) residue at position 166 wassubstituted to each of the other 19 amino acids using site-directedmutagenesis as previously described in the context of a pF4Ag vector(i.e., in the context of the wild-type OgLuc sequence SEQ ID NO: 1).These position 166 variants were then expressed in E. coli as previouslydescribed.

To create lysates, 50 μL 0.5× FASTBREAK™ Cell Lysis Reagent (PromegaCorp.) was added to 950 μl of induced cultures, and the mixturesincubated for 30 min at 22° C. For the analysis, 50 μL of lysate wasassayed in 50 μL of assay reagent (as previously described in Example22H) with either 100 μM PBI-3939, 30 μM native coelenterazine, or 22coelenterazine-h). Luminescence was measured as previously described(FIGS. 30A-C). FIGS. 30A-C show the relative activity of the N166mutants. Western analysis confirmed comparable expression of allvariants (data not shown).

B. The specific single amino acid substitutions, L27V, A33N, K43R, M75K,T39T, L72Q and F68D were assessed in the wild-type OgLuc or N166Rbackground. The single amino acid substitutions were generated viasite-directed mutagenesis as previously described, expressed in E. colias previously described, and luminescence measured using the assayreagent (previously described in Example 22H) with 22 μM nativecoelenterazine (FIG. 30D). Western analysis confirmed comparableexpression of all variants (data not shown).

Example 24 Deletion Variants

Deletions to the L27V variant were made as follows:

TABLE 16 Deletion # Deletion Made 27 Residues 1-27 and Val −1 52Residues 1-52 and Val −1 64 Residues 1-64 and Val −1 84 Residues 1-84and Val −1 19 Residues 65-83 23 Residues 65-87 23A1 Residues 65-87 +G64D

The N-terminus of the OgLuc variant L27V is methionine, valine andphenylalanine, i.e., MVF. For numbering purposes, the phenylalanine wasconsidered the first amino acid. “Val-1” indicates that the Valine in“MVF” was deleted. The methionine of “MVF” was included in thesedeletions. The L27 deletion variants were cloned in the pF4Ag vector andexpressed in E. coli KRX cells as previously described. Inductions andlysate preparations were performed as described Lysates were analyzedusing the assay reagent (previously described; 100 μM PBI-3939), andluminescence measured as previously described (FIG. 31). The datademonstrates that smaller fragments of the OgLuc variants can alsogenerate luminescence.

Example 25 Codon Optimization of OgLuc Variants

A. IV and 9B8

The IV and 9B8 OgLuc variants were used as templates for codonoptimization. The goals, as understood by those skilled in the art, weretwo-fold: 1) to remove known transcription factor binding sites, orother regulatory sequences, e.g., promoter modules, splicedonor/acceptor sites, splice silencers, Kozak sequences, and poly-Asignals, that could potentially interfere with the regulation orexpression of the OgLuc variants, and 2) to alter the DNA sequence (viasilent mutations that do not alter protein sequence) to eliminate rarelyused codons, and favor the most commonly used codons in cells of E.coli, human, other mammalian, or other eukaryotic organisms (Wada etal., Nucleic Acids Res., 18:2367 (1990)).

Two different optimized sequences for IV and 9B8, referred to as opt(aka optA) and optB, were designed for each variant. The first optimizedsequence, i.e., opt/optA for each variant, was designed by identifyingthe two best, i.e., most common, human codons for each site (see Table17) and then randomly picking one of the two for incorporation at eachsite. For the optB versions, the previous, codon-usage, optimizedversion, i.e., opt/optA, was used as a starting template, and each codonreplaced with the other of the two best human codons identified for thiscodon-optimization strategy. As an example, the leucine at position 3 ineither the IV or 9B8 sequence is encoded by the codon TTG. TTG is notone of the two most common codons for leucine in a human cell, andtherefore the codon was changed to the alternative, more common codonsfor leucine, CTC (opt/optA) or CTG (optB). This same process wasrepeated for all leucines in the sequence, and due to the random natureof the approach, a CTC codon could end up in optB and the CTG could endup in optA. Because of this two codon-usage approach to optimization,sequences opt/optA and opt B were maximally codon-distinct.

TABLE 17 Codons used in Codon Optimization Amino acid Choice#1 Choice#2Gly GGC GGG Glu GAG GAA Asp GAC GAT Val GTG GTC Ala GCC GCT Ser AGC TTCLys AAG AAA Asn AAC AAT Met ATG Ile ATC ATT Thr ACC ACA Trp TGG Cys TGCTGT Tyr TAC TAT Phe TTC TTT Arg CGG CGC Gln CAG CAA His CAC CAT Leu CTGCTC Pro CCC CCT

Each of the 4 sequences (IV opt, IV optB; 9B8 opt, 9B8 optB) were thenanalyzed (Genomatix Software, Germany) for the presence of transcriptionfactor binding sites or other regulatory sequences as described above,and these undesirable sequences were disrupted via silent nucleotidechanges. In some cases, where there were other non-rare codons for bothhuman and E. coli, the transcription factor binding sites or otherregulatory elements was removed by changing to one of these codons, eventhough they are not choice#1 or choice#2 (see Table 18). In cases, whereremoving a transcription factor binding site or other regulatory elementwould have involved introducing a rare codon, the transcription bindingsite (or other regulatory element) was usually not changed.

TABLE 18 Additional Codons used to Remove TranscriptionFactor Binding Sites and Other Regulatory Elements Amino Acid Choice #3Choice #4 Gly GGA GGT Val GTA GTT Ala GCG GCA Ser AGT TCA Thr ACG ACTLeu TTG CTT Pro CCG CCA

Codon optimized versions of IV (“IV opt” (SEQ ID NO: 22) and “IV optB”(SEQ ID NO: 23)) and 9B8 (“9B8 opt” (SEQ ID NO: 24) and “9B8 optB” (SEQID NO: 25)) were generated and cloned into pF4Ag by methods known in theart. HEK293 cells were plated in 96-well plates at 15,000 cells/well andgrown overnight at 37° C. The following day, the cells were transientlytransfected in 6 well replicates using TRANSIT®-LT1 Transfection Reagent(Minis Bio) with 100 ng of plasmid DNA encoding the codon optimizedversions in pF4Ag and grown overnight at 37° C. HEK293 cells were alsotransfected with either pGL4.13 (Luc2/SV40) (Paguio et al., “pGL4Vectors: A New Generation of Luciferase Reporter Vectors” Promega Notes,89:7-10 (2005)) or pGL4.73 (hRL/SV40) (Id.) to normalize for differencesin transfection efficiency. Ten ng/transfection or 10% of the total DNAtransfected was used. Media was removed, and cells were lysed with 100μL lysis buffer which contained 10 mM CDTA, 150 mM KCl, 10 mM DTT, 100mM HEPES, pH 7.0, 35 mM thiourea, and 0.5% TERGITOL® NP-9 (v/v) tocreate a lysate sample. Luminescence of the lysate sample was measuredon a TECAN® INFINITE® F500 luminometer as follows: for hRL and the OgLucvariants, 10 μL of the lysate sample was assayed for luminescence with50 μL of lysis buffer containing 20 μM of substrate (nativecoelenterazine for hRL and PBI-3939 for the OgLuc variants). For Luc2(SEQ ID NOs: 28 and 29), a firefly luciferase, 10 μL of lysate samplewas assayed for luminescence with 50 μL of BRIGHT-GLO™ Luciferase AssayReagent (Promega Corp.).

FIG. 32 shows a comparison of the luminescence measured for the lysatescontaining the codon optimized versions of the OgLuc variants comparedto hRL and Luc2. hRL and the OgLuc variants were normalized to pGL4.13and Luc2 was normalized to pGL4.73 using methods known in the art. Asshown in FIG. 32, Luc2 had approximately 14 fold higher luminescencethan hRL. The OgLuc variants had higher luminescence compared to Luc2and hRL. The codon optimized versions of IV (“IV opt” and “IV optB”) and9B8 (“9B8 opt”) showed increased luminescence compared to thenon-optimized versions.

As a result of this optimization, the “optioptA” versions expressedbetter in human HEK293 cells than their parental sequence, while the“optB” versions did not express as well in HEK293 cells compared to theparental sequence.

B. L27V

The L27V variant (SEQ ID NO: 88) was optimized to minimize theoccurrence of common vertebrate response elements (any transcriptionfactor binding site (TFBS) in the Genomatix database). Three differentoptimized versions of the L27V variant were created:

1. L27V01—version 1 (SEQ ID NO: 319)—Promoter modules and all otherundesired sequence elements (additional details below) were removed bynucleotide substitutions except for individual TFBSs.

2. L27V02—version 2—L27V01 was used as the starting, i.e., parental,sequence, and as many TFBSs were removed as possible using highstringency match criteria (A higher stringency involves a better matchto the binding site and will thus find fewer matches than a lowerstringency). There were two versions, A (SEQ ID NO: 322) & B ((SEQ IDNO: 318)), created for L27V02. These two versions were created byselecting different codons for each version to remove undesired sequenceelements. Both versions were analyzed by searching for TFBSs with lowerstringency.

3. L27V03—version 3 (SEQ ID NO: 325)—L27V02B (SEQ ID NO: 318) was usedas the starting sequence. Lower stringency TFBS matches were removedwhere possible. L27V03 was created to be very codon distinct fromL27V02A.

The following criteria were used to create the L27V optimized variants:

1. Codon usage: Preferably, the best two human codons were used for eachamino acid (as was done for the IV variant), and the use of rare humancodons (HS; coding for <10% of amino acids) was avoided (Table 19). Theuse of rare E. coli codons (EC) was used, if necessary, to removeundesired sequence elements.

TABLE 19 Amino Best Avoid acid Codons Codons A GCT A GCC C TGT C TGC DGAT D GAC E GAG E GAA F TTT F TTC G GGG G GGC H CAT H CAC I ATT

I ATC

K AAG K AAA L CTG TTA L CTC [HS] CTA [HS,EC] M ATG N AAT N AAC P CCT PCCC Q CAG Q CAA R CGG

R CGC

CGT [HS] S AGC TCG S TCC [HS] T ACA T ACC V GTG V GTC W TGG Y TAT Y TAC

2. Undesired sequence elements that were removed where possible

A. Restriction Enzyme (RE) Sites: RE sites were removed that would beuseful for cloning and should otherwise not be present in open readingframe (ORF).

B. Eukaryotic Sequence Elements: Splice donor and acceptor sites, splicesilencers, Kozak sequence and PolyA sequences in the (+) mRNA strandwere removed.

C. Vertebrate Promoter Modules (PM) (in Genomatix category: Vertebrate)were removed.

D. Vertebrate TFBS (in Genomatix categories: Vertebrate, general CorePromoter Elements, and miscellaneous other sequences) were removed wherepossible. This applied only to L27V optimized versions 2 and 3, but notto version 1.

E. E. coli Sequence Elements: E. coli promoters were removed.

F. mRNA Secondary Structure: Strong secondary structures (high mRNAfolding energy) near the 5′ end (Zuker, Nucleic Acid Res. 31(13):3406-3415 (2003)) and other strong hairpin structures were removed.

A sequence comparison, percent pair-wise sequence identity is providedin Table 20 (“( )” indicate number of nucleotide differences).

TABLE 20 L27V01 L27V02A L27V02B L27V03 L27V00 99% (3) 97% 97% 94% L27V0198% (12) 98% 94% (32) L27V02A 99% (4) 95% (26) L27V02B 96%

Example 26 Signal Stability of OgLuc Variants

A. 15C1, 9B and IV

The signal stability of 15C1 with PBI-3945 and 9B8 with PBI-3889 wasmeasured and compared to IV. E. coli containing plasmid DNA encoding15C1, 9B8, or IV were grown and induced as described previously in8-well replicates. Cells were lysed using a lysis buffer containing of300 mM HEPES pH 8.0, 0.3× Passive Lysis Buffer (“PLB”; Promega Corp.Cat. No. E194A), 0.3 mg/mL lysozyme, and 0.003 U/μL RQ1 DNase. Lysateswere diluted 1:1000 in lysis buffer and measured for luminescence usinga TECAN® INFINITE® F500 luminometer. Measurements were taken immediatelyafter the addition to 10 μL of diluted lysate sample of 50 μL of “Glo”0.5% TERGITOL assay buffer (“0.5% TERGITOL”), which contained 150 mMKCl, 1 mM CDTA, 10 mM DTT, 100 mM thiourea, 0.5% TERGITOL® NP-9 (v/v),and 20 μM of either novel coelenterazines PBI-3945 or PBI 3889.

The signal stability of the variants was determined by re-reading theplate every 30 seconds for a length of time after the addition of theassay buffer to the sample. The signal half-life was determined fromthese measurements using methods known in the art. The average signalhalf-life was compared between the variants and IV. Both 15C1 and 9B8had a signal half-life of at least 30 min (FIG. 33). Although 15C1,assayed with PBI-3945 had a higher luminescence at t=0, the signaldecayed more rapidly than variant 9B8 assayed with PBI-3889. At t=10min, luminescence for 15C1 with PBI-3945 and 9B8 with PBI-3889 wereequivalent.

B. 9B8 opt+K33N

The signal stability of the 9B8 opt+K33N variants was examined. E. colilysates containing the variants were prepared and analyzed as describedpreviously except the assay buffer contained 0.25% TERGITOL® NP-9 (v/v),100 mM MES pH 6.0, 1 mM CDTA, 150 mM KCl, 35 mM thiourea, 2 mM DTT, and20 μM PBI-3939. Table 22 shows the signal half-life in min of thevariants and indicates that the amino acid substitution L27V improvessignal stability.

TABLE 22 Signal stability of OgLuc variants in bacterial lysates samplesignal half life (min) 9B8 74 K33N 55 T39T, Y68D 87 T39T, L27V, K43R 139L27V, T39T, K43R, Y68D 114 T39T, K43R, Y68D 61 L27V, T39T, K43R, S66N124 L27V,K43R, Y68D 122 L27V, Y68D 139 L27V, K43R, S66N 124

The signal activity and stability of the L27V variant(9B8+K33N+L27V+T39T+K43R+Y68D; SEQ ID NO: 88 and 89) was measured andcompared to that of firefly (Luc2) and Renilla luciferases. The L27Vvariant, Luc2 and Renilla luciferases were fused to HALOTAG® andexpressed in E. coli. The luciferases were purified using HALOTAG® as apurification tag according to the manufacturer's protocol (pFN18A;HALOTAG® Protein Purification System). 10 μM of each purified luciferase(diluted in DMEM without phenol red containing 0.01% PRIONEX®) was thenmixed with an equal volume of an assay reagent (100 mM MES pH 6, 35 mMthiourea, 0.5% TERGITOL® NP-9 (v/v), 1 mM CDTA, 2 mM DTT, 150 mM KCl,and 100 μM PBI-3939 for the L27V variant; ONE-GLO™ Luciferase AssaySystem (Promega Corp.) for firefly luciferase; and RENILLA-GLO™Luciferase Assay System (Promega Corp.) for Renilla luciferase), andluminescence was monitored over time (3, 10, 20, 30, 45 and 60 min).FIGS. 34A-B demonstrates the high specific activity (FIG. 34A) andsignal stability (FIG. 34B) of the L27V variant when compared to fireflyand Renilla luciferase.

Example 27 Enzyme Kinetics of OgLuc Variants

A. IV, 15C1, 9B8, 9F6 and 9A3

Using methods known in the art, enzyme kinetic assays measuringluminescence were performed with the lysates of E. coli containing N andthe IV variants 15C1, 9B8, 9F6, and 9A3. Cells were induced, lysed, anddiluted as described in Example 26 except the lysis buffer had a pH of7.5. Two fold serial dilutions of PBI-3939 in the assay buffer describedpreviously in Example 26 were assayed with the diluted lysates. FIG. 35shows the Km and Vmax values calculated using a hyperbolic fit for IVand the variants 15C1, 9B8, 9F6, and 9A3. Variants 9B8 and 9F6 hadhigher Km values compared to IV while Km values for the other variantswere unchanged. Variants 15C1, 9B8, and 9F6 all had higher Vmax values,while 8A3 had a lower Vmax value compared to IV.

15C1, which had the highest luminescence with PBI-3945 contained theamino acid substitution K33N, indicating that K33N provided increasedluminescence. A 9B8 variant was generated to have this additionalsubstitution to provide improvement in luminescence for this variant.Additional variants of 9B8 and 9F6 were generated to have at least oneof amino acid substitutions K33N or V38I (“9B8+K33N+V38I” and“9F6+K33N”). Variant 1D6 was used to highlight the importance of aminoacid substitutions at positions 68, 72, and 75 for increasing lightoutput and stability. FIG. 36 shows the Km and Vmax values calculatedusing a hyperbolic fit for IV and the variants 9B8, 9B8+K33N+V38I, 9F6,9F6+K33N, and 1D6. While the actual Km values were different betweenFIGS. 35 and 36 for 9B8 and 9F6, the general trend between the variantswas consistent.

The enzyme kinetics, i.e., Vmax and Km values, were determined andcompared for the variants 9B8 opt and 9B8 opt+K33N as described aboveexcept the E. coli lysates were assayed with a buffer containing 1 mMCTDA, 150 mM KCl, 2 mM DTT, 100 mM MES pH 6.0, 35 mM thiourea, 0.25%TERGITOL® NP-9 (v/v), 10 mg/mL 2-hydroxypropyl-β-cyclodextrin, and 20 μMPBI-3939. Luminescence was measured on a TECAN® INFINITE® F500luminometer. As shown in FIG. 37, the Vmax and Km values for 9B8opt+K33N were higher than 9B8 opt, indicating that this clone isbrighter and has a lower affinity for substrate.

B. 9B8 OPT+K33N VARIANTS

The enzyme kinetics values were determined for the OgLuc variants asdescribed previously, except luminescence was measured using a GLOMAX®luminometer. Three replicates were used for each variant. Table 23 showsthe average Km and Vmax values with the standard deviation (“Km(+/−)”and “Vmax(+/−)” respectively) calculated using HYPER.EXE, Version 1.0.

TABLE 23 Vmax (RLU/0.5 sec) and Km (μM) values for OgLuc Variants sampleKm Km (+/−) Vmax Vmax (+/−) 9B8 7.7 2.0 86,000,000 14,000,000 K33N 12.53.0 110,000,000 17,000,000 1391, Y68D 7.9 1.8 74,000,000 10,000,000139T, L27V, K43R 21.4 5.4 150,000,000 28,000,000 L27V, 1391, K43R, Y68D13.9 2.9 190,000,000 28,000,000 139T, K43R, Y68D 10.5 2.8 140,000,00025,000,000 L27V, T391, K43R, S66N 16.3 4.8 130,000,000 28,000,000L27V,K43R, Y68D 13.7 4.3 130,000,000 28,000,000 L27V, Y68D 10.2 3.097,000,000 19,000,000 L27V K43R, S66N 20.0 6.2 130,000,000 30,000,000

Example 28 Protein Stability of OgLuc variants

As stability of the luciferase protein is another factor affectingluminescence, protein stability, i.e., thermal stability, of thevariants was determined.

A. 15C1, 9B8, 9F6, 8A3 and IV

Lysates of E. coli containing 15C1, 9B8, 9F6, 8A3 or IV and E. coliexpressing hRL (SEQ ID NO: 30 and 31) were prepared from inducedcultures as described previously. Lysate samples were diluted 1:1000with a buffer containing 10 mM HEPES pH 7.5 with 0.1% gelatin. Dilutedlysate (100 μL) samples, in replicate 96-well plates, were incubated at50° C. At different time points, plates were placed at −70° C. (minusseventy degrees Celsius). Prior to measuring the luminescence asdescribed previously, each plate was thawed at room temperature, i.e.,22° C., for 10 min. Samples (10 μL of each thawed sample) were assayedusing native coelenterazine as a substrate. Luminescence was measuredimmediately after addition of assay buffer for each time point plate.The half-life of the protein, which indicated protein stability, wascalculated from the luminescence data for each time point using methodsknown in the art.

Table 24 shows the protein stability of variants 15C1, 9B8, 9F6, and 8A3having half-lives in min (hrs) of 630.1 (10.5), 346.6 (5.8), 770.2(12.8) and 65.4 (1.1), respectively. In comparison, hRL had a half-lifeof 9.6 min, while IV had a half-life of 27.2 min. Table 24 also showsthat at 4 hrs, 79%, 61%, and 80% of 15C1, 9B8, and 9F6, respectively,remained active.

TABLE 24 Protein Stability of OgLuc Variants at 50° C. Sample ½ life(min) ½ life (hrs) % remaining at t = 4 hrs Renilla 9.6 IV 27.2 15C1630.1 10.5 79% 9B8 346.6 5.8 61% 9F6 770.2 12.8 80% 8A3 65.4 1.1

B. 1D6, 9B8, 9B8+K33N+V381, 9F6, 9F6+K33N, and IV

Lysates of E. coli containing 1D6, 9B8, 9B8+K33N+V38I, 9F6, 9F6+K33N, orIV were prepared from induced cultures and assayed for luminescence asdescribed previously. Protein stability, i.e., thermal stability of thelysates, was assayed as described above in this Example. FIG. 38 showsthe half-life in minutes (min) of the variants at 50° C., and theluminescence of the sample measured at the start of the incubationperiod, i.e., t=0, using native coelenterazine as a substrate. Thedifference between variant 9B8+33+38 and 9F6 was one amino acidsubstitution, L27V, indicating that this amino acid substitutionincreased stability. The addition of “activity/expression” substitutionsin positions 68, 72, and 75 increased stability. FIG. 38 shows K33Nprovided greater thermal stability to variant 9F6 and that variant 9B8had greater light output and stability than variant 1D6. The differencebetween these two variants, i.e., 9B8 contains additional amino acidsubstitutions F68Y, L72Q, and M75K, indicated the importance of thesethree substitutions.

In addition to thermal stability, structural integrity determined byexpression, stability, and solubility can also affect luminescence. As away to further test the structural integrity of the improved variants,KRX E. coli harboring pF4Ag-based (i.e., no HT7) OgLuc variants N166R(previously described in U.S. application Ser. No. 12/773,002 (U.S.Published Application No. 2010/0281552)), Cl⁺ A4E, IV, 9B8, and 9F6 weregrown at 37° C. in Luria broth (LB) to an OD₆₀₀=0.6 and then induced foroverexpression by the addition of rhamnose (0.2% final concentration).Duplicate induced cultures were then grown at either 25 or 37° C. for 17hrs at which time total (T) and soluble (S) fractions were prepared andanalyzed by SDS-PAGE using SIMPLYBLUE™ SafeStain (Invitrogen) to stainthe gels (FIGS. 39A-B). hRL and Luc2 were used as controls.

The OgLuc variants, hRL and Luc2 expressed well and were soluble whenthe induction occurred at 25° C. (FIG. 39A; note the approximately 19kDa dark band in the “soluble” fraction for the OgLuc variants,excluding the N166R variant, and the approximately 36 and 64 kDa bandsin the “soluble” fraction for hRL and Luc2, respectively). In contrast,although C1+A4E, IV, 9B8, and 9F6 expressed well at 37° C.(significantly better than hRL or Luc2, as shown in the “total”fraction), only the 9B8 and 9F6 variants were soluble when the elevatedinduction temperature was employed (see FIG. 39B; note the approximately19 kDa dark band in the “soluble” fraction for 9B8 and 9F6). Theseresults tracked with the thermal stability data shown in Table 24 andFIG. 38.

C. 9B8 OPT and 9B8 OPT+K33N

The thermal stability of the variants 9B8 opt and 9B8 opt+K33N wascompared. E. coli lysates containing the variant 9B8 opt or 9B8 opt+K33Nwere prepared and analyzed as described previously with the followingexceptions: Lysates were diluted 1:100 in the lysis buffer describedpreviously and replicate diluted lysates were incubated at 60° C. in athermocycler. Aliquots were removed at different time-points and placedon dry ice to freeze the samples. Frozen lysates were thawed at 22° C.and assayed with a buffer containing 20 mM CDTA, 150 mM KCl, 10 mM DTT,20 μM PBI-3939, 100 mM HEPES pH 7.0, 35 mM thiourea, and 0.1% TERGITOL®NP-9 (v/v). Luminescence was measured on a GLOMAX® luminometer (PromegaCorp.). FIG. 40A shows the light output time course of the naturallogarithm (ln) value of luminescence measured in RLU over time in min.As shown in FIG. 40B, 9B8 opt+K33N had a half-life at 60° C. of 6.8 hrs,which was longer than the 5.7 hrs half-life of 9B8 opt.

Table 25 shows the thermal stability at 60° C. (“T_(1/2) (60° C.)”) of9B8 opt and 9B8 opt+K33N, and the luminescence (“RLU”) data at the startof the incubation period (i.e., t=0). 9B8 opt+K33N was more stable andapproximately 1.8-fold brighter than 9B8 opt, indicating that the aminoacid substitution K33N provided both greater light output and higherthermal stability.

TABLE 25 Thermal Stability and Luminescence Data for 9B8 opt and 9B8opt + K33N Variant T_(1/2)(60° C.) RLU 9B8 opt 5.7 hrs 23,283,252,0009B8 opt + K33N 6.8 hrs 42,278,732,000

D. 9B8+K33N Variants

The thermal stability of the variants at 60° C. was examined asdescribed above, except the assay buffer contained 100 mM MES pH 6.0instead of HEPES. Table 26 and FIG. 41 shows the half-life in hrs of thevariants at 60° C. The data indicates that the amino acid substitutionL27V improves thermal stability.

TABLE 26 Thermal stability of OgLuc variants at 60° C. Sample ½ lifehours 9B8  5.1 K33N  6.7 T39T, Y68D 16.3 T39T, L27V, K43R 11.8 L27V,T39T, K43R, Y68D 21.7 T39T, K43R, Y68D 15.2 L27V, T39T, K43R, S66N 11.8L27V,K43R, Y68D 23.2 L27V, Y68D 28.5 L27V, K43R, S66N 10.7

The variants 9B8 and V2 (9B8+K33N+T39T+K43R+Y68D) were also screened inHEK293 cells to determine their stability. The variants were cloned intopF4Ag and transfected into HEK293 cells (15,000 cells/well) aspreviously described. After transfection, the cells were lysed in assayreagent (as previously described; no PBI-3939), and luminescencemeasured using the assay reagent with 20 μM PBI-3939. 9B8 had ahalf-life of 5.2 hrs while V2 had a half-life of 16.8 hrs. This isconsistent with the half-life seen for these variants in E. coli (Table26).

E. L27V Variant

The activity of the L27V variant (9B8+K33N+L27V+T39T+K43R+Y68D) wasassessed at various pHs and different salt conditions. 9B8 and 9B8+K33Nwere previously shown to have similar stability at pH 6 and pH 7 (datanot shown). For assessing activity at various salt conditions, 50 μL ofassay buffer with 20 μM PBI-3939 and varying amounts of KCl or NaCl wasmixed with 50 μL of HEK293 cells transiently transfected with L27V(pF4Ag). Luminescence was measured, and the percent activity (the ratioof luminescence to no salt) determined (FIG. 42B). For assessingactivity in various pHs, a reagent was made containing 100 mM citrate,100 mM MES, 100 mM PIPES, 100 mM HEPES, 100 mM TAPS, 0.5% TERGITOL® NP-9(v/v), 0.05% MAZU® DF 204, 1 mM CDTA, and 1 mM DTT titrated to variouspH values. 362 μM L27V in assay reagent was mixed with substrate 100 μMPBI-3939 and luminescence was measured. (FIG. 42A).

Example 29 Gel Filtration Chromatographic Analysis of OgLuc Variants

A. C1+A4E and 9B8

Gel filtration analysis was used to verify the expected molecular weightof the purified OgLuc proteins based on the theoretical values andconsequently to determine their oligomeric state. A comparison betweenthe relative hydrodynamic volume of the OgLuc variants C1+A4E and 9B8was made by gel filtration chromatography. For this analysis, thenucleotide sequence for the OgLuc variants, C1+A4E and 9B8, were clonedinto a HQ-Tagged FLEXI® Vector (Promega Corp.) to create a HQHQHQN-terminally tagged protein that was overexpressed in E. coli KRX cells.The overexpressed proteins were purified using the HISLINK™ ProteinPurification System (Promega Corp.) according to manufacturer'sinstructions. Samples of each individual standard and sample proteinwere analyzed by gel filtration chromatography, which was performed at24° C. on an Agilent 1200 HPLC, using a Superdex 200 5/150 GL column (GEHealthcare) with a flow rate of 0.25 mL/min (FIGS. 43A-B). The mobilephase (i.e., running buffer) consisted of 50 mM Tris and 150 mM NaCl, pH7.5. Protein elution was monitored at 214 and 280 nm. A standardcalibration curve was generated using: 1) Ovalbumin, 43 kDa (GEHealthcare), 2) Carbonic Anhydrase, 29 kDa (Sigma) and 3) Myoglobin, 17kDa (Horse Heart, Sigma). The molecular weights of the purified proteinswere calculated directly from the calibration curve.

The relative elution of the proteins observed with this column wasOvalbumin at 7.98 min, Carbonic Anhydrase at 8.65 min, 9B8 at 8.97 min,and Myoglobin at 9.06 min (FIGS. 43A-B). As shown in FIG. 43B, 9B8eluted as a 21 kDa protein (predicted MW is approximately 19 kDa)indicating that the 9B8 variant existed as a monomer, whereas C1+A4Eeluted at approximately 4.3 min (FIG. 43A), indicating that C1+A4E wasexpressed and exists as multimer, e.g., possibly as a tetrameric complexor something larger.

B. L27V Variant

To demonstrate that the OgLuc variant L27V exists in a monomeric state,gel filtration analysis was used to verify the expected molecular weightof the purified L27V protein based on the theoretical value, andconsequently to determine its oligomeric state. The relativehydrodynamic volume of the L27V variant was made by gel filtrationchromatography. For this analysis, the nucleotide sequence for the L27Vvariant was cloned into a HaloTag® vector pFN18A (Promega Corp.) tocreate a HaloTag®-terminally tagged protein that was overexpressed in E.coli KRX cells (Promega Corp.). The overexpressed protein was purifiedusing the HaloTag® Protein Purification System (Promega Corp.) accordingto manufacturer's instructions. Samples of each individual standard andsample protein were analyzed by gel filtration chromatography performedat 24° C. on an Agilent 1200 HPLC using a Superdex 200 5/150 GL column(GE Healthcare) with a flow rate of 0.25 mL/min (FIG. 56). The mobilephase (i.e., running buffer) consisted of 50 mM Tris and 150 mM NaCl, pH7.5. Protein elution was monitored at 214 and 280 nm. A standardcalibration curve was generated using: 1) Ovalbumin, 43 kDa (GEHealthcare), 2) Myoglobin, 17 kDa (Horse Heart, Sigma), and 3)Ribonuclease, 14 kDa (Bovine pancreas, GE Healthcare). As shown in FIG.44, the L27V variant eluted as a 24 kDa protein (predicted MW isapproximately 19 kDa) indicating that it existed as a monomer.

Example 30 Protein Expression Levels of OgLuc Variants

A. IV, 8A3, 8F2, 9B8, 9F6 and 15C1

Normalizing for protein expression provides information about potentialdifferences in specific activity. To provide a means for quantifyingprotein expression, OgLuc variants were cloned into a pF4Ag vectorcontaining a C-terminal HT7 to generate OgLuc variant-HT7 fusionproteins as described previously. The following fusion proteins weregenerated: IV-HT7 (SEQ ID NOs: 48 and 49), 8A3-HT7 (SEQ ID NOs: 34 and35), 8F2-HT7 (SEQ ID NOs: 50 and 51), 9B8-HT7 (SEQ ID NOs: 36 and 37),9F6-HT7 (SEQ ID NOs: 38 and 39), and 15C1-HT7 (SEQ ID NOs: 52 and 53).E. coli containing the OgLuc variant-HT7 fusions were grown and inducedas described previously. 900 μL of cell culture was lysed with 100 μL of10× FASTBREAK™ Cell Lysis Reagent (Promega Corp.). HALOTAG® TMR-ligand(Promega Corp.) was added to each bacterial lysate sample to obtain afinal concentration of 0.5 μM. Bacterial lysates were incubated with theHALOTAG® TMR-ligand for 30 min at room temperature according tomanufacture's instructions. 10 μL of each sample was diluted 1:1 with 1×FASTBREAK™, i.e., 10 μL sample to 10 μL 1× FASTBREAK™. 15 μL of thelysate and 15 μL of the 1:1 dilution for each sample were analyzed bySDS PAGE. The labeled fusion proteins were resolved by SDS-PAGE, stainedwith SIMPLYBLUE™ SafeStain (FIG. 45A) and fluorimaged (GE HealthcareTyphoon). Bands were quantitated using Imagequant software (GEHealthcare). FIG. 45B shows the band volume measured from FIG. 45A forIV-H1′7 (“IV”), 15C1-HT7 (“15C1”), 9B8-HT7 (“9B8”), 9F6-HT7 (“9F6”), and8F2-HT7 (“8F2”), normalized to IV-HT7. The data shows that the IVvariants expressed well compared to IV.

B. 9B8 opt, V2 and L27V

The expression levels and solubility of 9B8 opt, V2 and L27V werecompared. These three variants, in the context of a pF4Ag background,were used to transform E. coli KRX cells. The resulting clones were usedfor an expression experiment where single colonies were grown overnightat 30° C., diluted 1:100 in LB, grown to an OD₆₀₀ approximately 0.5, andthen induced with 0.2% rhamnose for 18 hrs at 25° C. Cells were thenincubated for 30 min at room temperature in the presence of 0.5×FASTBREAK™ Lysis Reagent (Promega Corp.), and the resulting lysatesstored at −20° C. Following a slow-thaw on ice, soluble fractions wereprepared by high-speed centrifugation for 10 min at 4° C. Crude total(T) and soluble (S) fractions were then analyzed for expression levelsusing SDS-PAGE+ Simply blue staining (FIG. 46A) as well as by measuringluminescence (FIG. 46B). For luminescence measurement, 50 μL of solublelysates in 96-well microtiter plates were mixed with 50 μL assay reagent(previously described; 40 μM PBI-3939), and luminescence measured usinga TECAN® INFINITE® F500 multi-detection plate reader. These resultsindicate that the ranking for these three variants, in terms of theirexpression levels and solubility, is L27V>V2>9B8opt.

Example 31 Brightness of OgLuc Variants Expressed in Mammalian Cells

A. IV and 9B8

The IV and 9B8 variant in pF4Ag vector (i.e., no HT7) were evaluated forbrightness in HEK293 cells. hRL was used as a control. Briefly, HEK293cells, plated at 15,000 cells/well in a 96-well plate, were transientlytransfected using TRANSIT®-LT1 with plasmid DNAs encoding the variousvariants and/or control sequences. Cells were grown, lysed, and treatedas described in Example 25. Cells were co-transfected with pGL4.13(Promega Corp.) as a transfection control (10 ng/transfection or 10% ofthe total DNA transfected was used). Luminescence was measured asdescribed previously using native coelenterazine as a substrate for hRLor PBI-3939 as a substrate for the OgLuc variants. The OgLuc variantdata was corrected for transfection efficiency using Luc2 luminescence(i.e., measuring luminescence after the addition of luciferinsubstrate). The OgLuc variants IV and 9B8 had greater luminescencecompared to hRL (“Renilla”) (FIG. 47).

For comparison of brightness on a per mole basis in mammalian cells, theC-terminal HT7 fusion protein of variant 9B8 (“pF4Ag-OgLuc-9B8-HT7”)described in Example 30 was analyzed and compared with C-terminalHT7-hRL fusion protein (“pF4Ag-Renilla-HT7”) and C-terminal HT7-Luc2fusion protein (“pF4Ag-Luc2-HT7”). HEK293 cells (15,000) were plated andgrown overnight at 37° C. These cells were transfected with 100 ng ofDNA from pF4Ag-Renilla-HT7, pF4Ag-Luc2-HT7, or pF4Ag-OgLuc-9B8-HT7 andgrown overnight at 37° C. Media was removed and cells were lysed asdescribed previously. 10 μL of each sample was assayed for luminescence(RLU) with 50 μL BRIGHT-GLO™ for Luc2, 50 μL of 20 μM nativecoelenterazine for hRL, and 50 μL of 20 μM PBI-3939 for variant 9B8.

The lysates from 6 wells were pooled and labeled with HALOTAG®TMR-ligand as described in Example 30. The labeled fusion proteins wereresolved by SDS-PAGE and fluorimaged (GE Healthcare Typhoon). The banddensities were determined to quantitate the relative number of molespresent for each luciferase enzyme and the RLU value for each sample wasnormalized by the calculated band density to normalize expression levelsof each protein, i.e., RLUs normalized using TMR label quantitation(FIG. 48). On a mole-to-mole basis, the 9B8 variant was approximately15-fold brighter than Luc2 and >100-fold brighter than hRL. This datarepresented differences in specific activity.

B. 9B8 opt and 9B8 opt+K33N

The brightness of the variants 9B8 opt and 9B8 opt+K33N expressed inHEK293 cells was measured and compared as described for the variantswithout the HT7 in Example 31. 30 and 100 ng of plasmid DNA containingthe variant DNA was used to transfect HEK293 cells. Cells were grown andinduced as described in Example 31 except the cells were lysed with alysis buffer containing 1 mM CTDA, 150 mM KCl, 2 mM DTT, 100 mM MES pH6.0, 35 mM thiourea, 0.25% TERGITOL® NP-9 (v/v), and 10 mg/mL2-hydroxypropyl-β-cyclodextrin. The lysates were assayed with lysisbuffer containing 20 μM PBI-3939 and luminescence was measured on aTECAN® GENIOS™ Pro luminometer. As shown in FIG. 49, 9B8 opt+K33N hadgreater luminescence compared to 9B8 opt in HEK293 cells, which trackswith the bacterial expression data in Table 25 and FIG. 29.

C. 9B8+K33N Variants

The brightness of the variants expressed in HEK293 and NIH3T3 cells wasmeasured as described previously. The luminescence of the variants wasnormalized to the luminescence generated by 9B8 opt (Table 27).

TABLE 27 Increase in Luminescence generated by OgLuc combinationvariants in NIH3T3 and HEK293 cells Sample HEK293 NIH3T3 9B8 1.0 1.0K33N 1.8 1.5 T39T, Y68D 1.9 1.5 T39T, L27V, K43R 1.3 0.9 L27V, T39T,K43R, Y68D 1.6 1.6 T39T, K43R, Y68D 1.9 1.9 L27V, T39T, K43R, S66N 1.31.2 L27V, K43R, Y68D 1.6 1.5 L27V, Y68D 1.7 1.4 L27V, K43R, S66N 1.2 1.0

D. L27V

A comparison of the luminescence of the L27V variant to fireflyluciferase alone and as a fusion was performed. HEK293 and HeLa cellswere plated at 15,000 and 10,000 cells/well, respectively, into wells of12-well plates and incubated overnight at 37° C., 5% CO₂. The cells werethen transfected with serial dilutions of pF4Ag containing L27V or Luc2.20 ng of pGL4.13 (Promega Corp.) was co-transfected with L27V, and 20 ngof pGL4.73 (Promega Corp.) was co-transfected with Luc2 to act ascarrier DNA for lower dilutions of the L27V or Luc2 plasmid DNA. Theplasmid DNA was then transfected into the cells (6 replicates for eachdilution for each cell type) using TRANSIT®-LTI transfection reagentaccording to the manufacturer's instructions. The cells were thenincubated for 24 hrs at 37° C., 5% CO₂.

After transfection, the media was removed from the cells, and 100 μL PBSwith 0.5% TERGITOL® NP-9 (v/v) added and shaken for 10 min at roomtemperature. 10 μL of each cell lysate was assayed using ONE-GLO™Luciferase Assay System (Promega Corp.; Luc2) or assay reagent (Example22H with 20 μM PBI-3939; OgLuc). Luminescence was measured as previouslydescribed for the HEK293 (FIG. 50A) and HeLa cells (FIG. 50B).

Comparison of L27V and Luc2 as fusion partners was performed asdescribed above. L27V and Luc2 were fused to HALOTAG® protein in pF4Ag.FIGS. 50C-D show the luminescence measured with the different fusions inHEK293 (FIG. 50C) and HeLa cells (FIG. 50D).

In addition to measuring luminescence, protein expression was alsoanalyzed. The transfection was performed as described above. Aftertransfection, the media was removed from the cells, and the cells washedin 1×PBS. 100 μL 0.1× Mammalian Lysis Buffer (Promega Corp.) containing1 μM HALOTAG®TMR ligand (Promega Corp.) and 20 U DNase I was added, andthe cells incubated with slow shaking for 45 min at room temperature.The cell samples were then frozen at −20° C. For protein analysis, 32.5μL 4×SDS loading dye was added to each sample, and the samples heated at95° C. for 2 min. 10 μL of sample was then loaded onto an SDS-PAGE geland imaged on a Typhoon Scanner as previously described (FIG. 50E).

Example 32 Brightness of Purified OgLuc Variant Compared to FireflyLuciferase

The 9B8 OgLuc variant was overexpressed and purified as described inExample 33. Reactions between diluted enzyme and substrate wereperformed using the following 2× buffer/assay reagent: 100 mM MES pH6.0, 1 mM CDTA, 150 mM KCl, 35 mM thiourea, 2 mM DTT, 0.25% TERGITOL®NP-9 (v/v), 0.025% MAZU® DF 204, 10 mg/mL 2-hydroxy-D-cyclodextrin, and20 μM PBI-3939. The final assay concentrations of purified enzyme andsubstrate were 0.5 μM and 10 μM, respectively. In parallel, reactionsbetween diluted purified firefly luciferase (i.e., QUANTILUM®Recombinant Luciferase (Promega Corp.)) and luciferin were analyzed. Theassay buffer/reagent for the firefly luciferase reaction wasBRIGHT-GLO™, and the final assay concentrations were 0.5 μM enzyme and500 μM luciferin. As the buffers/reagents for each reaction were knownto provide “glow” kinetics, a 15 min time point was used to collectluminescence data. The results from this experiment showed that 9B8 optusing PBI-3939 (19,200 RLU) was approximately 8-fold brighter thanQUANTILUM® Recombinant Luciferase with BRIGHT-GLO™ (2,300 RLU).

Example 33 Inhibition Analysis

To determine the susceptibility of the OgLuc variants to off-targetinteractions, the activity of the 9B8 and L27V variants was screenedagainst a LOPAC (library of pharmacologically active compounds) library.A LOPAC 1280 library (Sigma) was prepared by diluting the compounds to 1mM in DMSO. On the day of the assay, the compounds were diluted to 20 μMin 1×PBS, and 10 μL transferred to a 96-well, white plate. To each well,10 μL of purified 9B8, L27V or firefly luciferase (Luc2) enzyme diluted10⁻⁴ in Glo Lysis Buffer (Promega Corp.) was added and incubated at roomtemperature for 2 min. To the samples, 20 μL assay reagent (1 mM CDTA,150 mM KCl, 2 mM DTT, 100 mM MES pH 6.0, 35 mM Thiourea, 0.5% TERGITOL®NP-9 (v/v) and 60 μM PBI-3939) was added, incubated for 3 min, andluminescence measured on a TECAN® GENIOS™ Pro Luminometer. For assayingfirefly luciferase, the BRIGHT-GLO™ Assay reagent (Promega Corp.) wasused according to the manufacturer's protocol. As a negative control, 8wells of each plate contained 1×PBS+2% glycerol. As a positive control,8 wells of each plate contained 2 mM Suramin in 2% DMSO or 2 mMluciferase inhibitor 1 in 2% DMSO (Calbiochem). Suramin was identifiedin the preliminary screen of the LOPAC library (i.e., the LOPAC librarywas screened using the 9B8 variant with a lower substrate concentrationof 20 μM) to be an inhibitor of OgLuc.

The results in FIG. 51 indicate a general low frequency of off-targetinteractions between the compounds in the LOPAC library and L27V. Thissuggests a potential use for L27V as a screening tool for largelibraries of diverse chemicals and therapeutic candidates, includinglive cell-based formats (e.g., high-throughput screening).

To further examine inhibition resistance, purified 9B8 and L27V werescreened against various concentrations of Suramin (Sigma S-2671) andTyrphostin AG 835 (“Tyr ag 835”) (Sigma T-5568) (FIGS. 52A-C). FIGS.52E-D show the chemical structures for Suramin and Tyr ag 835,respectively. Purified 9B8 and L27V were prepared as described above.Serial dilutions (0, 2 μM, 6 μM, 20 μM, 60 μM, 200 μM and 2 mM) of theinhibitors were prepared in 1×PBS with 2% DMSO. To wells of a 96-well,white assay plate, 10 μL of diluted enzyme and 10 μL of dilutedinhibitor were added and incubated at room temperature for 2 min. 20 μLassay reagent (described above) was added, and luminescence measured ona GLOMAX®-96 luminometer (FIGS. 52A-C). FIGS. 52A-B show the doseresponse curves of 9B8 and L27V to Suramin (FIG. 52A) and Tyr ag 835(FIG. 52B). FIG. 52C shows the half maximal inhibitory concentration(IC₅₀) of Suramin and Tyr ag 835 for 9B8 and L27V. The data indicatesthat L27V is a robust reporter that could be used as a screening toolfor large libraries of diverse chemicals and/or therapeutic candidates.

Example 34 Resistance to Non-Specific Protein Interactions

1. Purified 9B8 and L27V enzyme were serial diluted in 1:10 in buffer(1×PBS, 1 mM DTT, and 0.005% IGEPAL® CA-630) with or without 0.5 mg/mLBSA (4 sets of each dilution) to 200 μL into PCR strip tubes. Thesamples were incubated at 60° C. wherein at 0, 2, 4, and 6 hrs one setof dilutions for each variant was transferred to −70° C.

To analyze activity, the samples were thawed to room temperature in awater bath. 50 μL assay reagent (as previously described with 100 μMPBI-3939) was added, and luminescence measured for each minute for 30min on a TECAN® INFINITE® F500 plate reader. Activity was calculatedusing the average luminescence of the 1×10⁶ and 1×10⁷ dilutions (FIG.53).

2. To demonstrate the reactivity of the OgLuc variants to plastic,purified 9B8 and L27V were exposed to polystyrene plates, and theiractivity measured.

50 μL purified 9B8 (45.3 μM) and L27V (85.9 μM) in DMEM without phenolred with 0.1% PRIONEX® was placed into wells of a 96-well, polystyrenemicrotiter plate at 60, 40, 20 and 0 min. To the samples, 50 μL assayreagent (described above) containing 20 M PBI-3939 was added andincubated for 5 min at room temperature. Luminescence was measured aspreviously described, and percent activity determined (FIG. 54; ratio ofluminescence to time 0).

Example 35 Post Translational Modification

To determine if the OgLuc variants undergo any post translationmodifications when expressed in mammalian cells, the 9B8 and L27Vvariants were expressed in both mammalian cells and E. coli and analyzedvia mass spectrometry (MS).

9B8 and L27V variants were expressed as N-terminal HALOTAG® fusions(pFN18K for E. coli; pFN21K for HEK293 cells) in HEK293 and E. coli KRX(Promega Corp.) cells and purified using the HALOTAG® ProteinPurification System (Promega Corp.) according to the manufacture'sinstructions. Approximately 5 pmols of purified enzyme was analyzed viaLC/MS using a C4 column (Waters Xbridge BEH300, 3.5 μm) interfaced to anLTQ Orbitrap Velos mass spectrometer (Thermo Scientific). Data wasacquired from 600-2000 m/z using the LTQ for detection and processedusing the MagTran v1.03 software (Zhang et al., J. Am. Soc. MassSpectrom., 9:225-233 (1998)). Both purified enzymes had anexperimentally determined mass of 19,666 Da, compared to a calculatedmass of an un-modified OgLuc variant, i.e., absent of any posttranslational modifications, of 19,665 Da.

Example 36 Evaluation of OgLuc Variants as a Transcriptional Reporter

A. IV

The use of the OgLuc variants as a transcriptional reporter wasexamined. To generate a transcriptional reporter of cAMP, hRL and IVwere sub-cloned using methods known in the art into a modified pGL4vector (Promega Corp.) containing a barnase sequence, which was replacedby the DNA fragment of interest. The leader sequence of the modifiedpGL4 contained a minimal promoter and a cAMP-response element (CRE; SEQID NO: 96), so that upon stimulation with a cAMP agonist such asforskolin (FSK), cells accumulating cAMP activated the reporter andgenerated luminescence. In this experiment, 2 ng DNA of either the hRLor IV transcriptional reporter construct was used to transfect HEK293cells as described in Example 25. At 24 hrs post transfection, the cellswere treated with 100 μM FSK. Cells that were not treated with FSK wereused as a control. After 6 hrs, a reporter reagent was added to treatedand control cells. For hRL, the reporter reagent was Renilla-Glo™reagent (Promega Corp.). For IV, the reporter reagent contained 1 mMCDTA pH 5.5, 150 mM KCl, 10 mM DTI', 0.5% TERGITOL® NP-9 (v/v), 20 μMcoelenterazine-h, and 150 mM thiourea. After 10 min, luminescence wasread on a Varioskan® Flash (Thermo Scientific).

FIG. 55 shows the normalized luminescence of HEK293 cells containing thehRL (“Renilla”) or IV transcriptional reporter treated (“+FSK”) or nottreated (“−FSK”) with FSK. The response, i.e., fold-induction orfold-increase (“FOLD”) in luminescence was determined by dividing theluminescence from the treated cells (+FSK) with the luminescence fromthe control cells (−FSK). As shown in FIG. 55, the response for hRL was<50, while for IV it was >300, demonstrating the use of IV as atranscriptional reporter.

B. 9B8 and 9B8 opt

The use of variants 9B8 and 9B8 opt as a transcriptional reporter wasalso examined and compared to hRL and Luc2 transcriptional reporters aspreviously described for the IV transcriptional reporter with thefollowing modifications. Transcriptional reporters of cAMP containingeither variants 9B8 or 9B8 opt were generated as described above. After6 hrs of FSK induction, the media was removed from the cells andreplaced with 100 μL of the lysis buffer described in Example 25creating a lysate. The lysate of transfected cells treated with orwithout FSK were assayed for luminescence as described in Example 25. 10μL of the Luc2 lysate was assayed with 50 μL of BRIGHT-GLO™ LuciferaseAssay Reagent. 10 μL of the hRL lysate was assayed with 50 μL of lysisbuffer containing 20 μM native coelenterazine. 10 μL of the variants 9B8and 9B8 opt lysates were assayed with 50 μL of lysis buffer containing20 μM PBI-3939.

FIG. 56 shows the normalized luminescence of HEK293 cells containing the9B8, 9B8 opt, hRL, or Luc2 transcriptional reporter treated (“induced”)or not treated (“basal”) with FSK. The response, i.e., fold-induction orfold-increase (“fold”) in luminescence, was determined by dividing theinduced luminescence by the basal luminescence (FIG. 56). Although thefold induction values are similar for each of the reporters except Luc2,the luminescence generated by the induced 9B8 opt transcriptionalreporter was approximately 2.5 logs higher than the induced Renillatranscriptional reporter and approximately 1.5 logs higher than the Luc2transcriptional reporter. FIG. 56 demonstrated the use of 9B8 and 9B8opt as transcriptional reporters.

C. 9B8 opt and 9B8 opt+K33N

The variants 9B8 opt and 9B8 opt+K33N were compared in a lytictranscriptional reporter assay. The variant 9B8 opt+K33N was clonedusing methods known in the art into a pGL4.29 vector (Promega Corp.),which contains a cyclic AMP response element (CRE). The 9B8 opt+K33Ntranscriptional reporter was tested and compared to the 9B8 opttranscriptional reporter as described above in HEK293 cells. 30 and 100ng of plasmid DNA containing the transcriptional reporter versions ofthe variants were used to transfect HEK293 cells. The cells were inducedwith FSK for 5 hrs prior to measurement for luminescence. Cells werelysed with a lysis buffer containing 1 mM CTDA, 150 mM KCl, 2 mM DTT,100 mM MES pH 6.0, 35 mM thiourea, 0.25% TERGITOL® NP-9 (v/v), and 10mg/mL 2-hydroxypropyl-β-cyclodextrin. Luminescence was measured on aTECAN® GENIOS™ Pro luminometer. The lysate was assayed with the lysisbuffer containing 20 μM PBI-3939. FIG. 57 shows the normalizedluminescence (transfection corrected) of HEK293 cells expressing the 9B8opt or 9B8 opt+K33N transcriptional reporter construct treated(“Induced”) or not treated (“Basal”) with FSK. As shown in FIG. 57, thefold-induction for 9B8 opt was 360 when 30 ng of DNA was used fortransfection and 109 when 100 ng was used for transfection, while thefold-induction for 9B8 opt+K33N was 275 and 147, respectively. Whenhigher amounts of DNA were used for transfection, K33N provided agreater response.

D. L27V

1. L27V was cloned into a reporter vector as described in C of thisExample containing a CRE, NFkB or HSE (Heat shock element) responseelement. Reporter constructs were then transfected into HEK293 cells orHeLa cells as previously described. The cells were then induced usingFSK for CRE, TNFα for NFkB or 17-AAG for HSE. Luminescence was measuredas previously described using the assay reagent with 20 μM PBI-3939(FIGS. 58A-C). The reporter constructs were all validated in HEK293,HeLa, NIH3T3, U20S and Jurkat cell lines (data not shown).

2. L27V02 and L27V02P (containing a PEST sequence; SEQ ID NO: 323) werecloned into a reporter vector (pGL4.32 based) as described in C of thisExample. Other OgLuc variants containing a PEST sequence includeL27V01-PEST00 and L27V03-PEST02 (SEQ ID NOs: 320 and 326, respectively).The reporter construct was then transfected into HEK293 cells aspreviously described. The cells were then induced using FSK, andluminescence was measured as previously described using the assayreagent with 20 μM PBI-3939 (FIGS. 59A-B). Various other reporterconstructs were also created and tested in various cell lines (FIG.59C). FIG. 59A shows the full dose response for the CRE system in HEK293cells. FIG. 59B summarizes FIG. 59B. FIG. 59C summarizes the data inFIGS. 59A-B and shows the same type of data for the NFkB responseelement. Both CRE and NFkB report constructs were examined in HEK293,HeLa, HepG2, Jurkat, ME180, HCT116, and U20S cell lines.

3. HEK293 cells (0.9×10⁶ cells in a T25 flask) were transfected withpNFkB-L27V secretion construct (SEQ ID NOS: 463 & 464; wherein the IL-6secretion sequence (SEQ ID NOs: 461 and 462) replaced the native OgLucsecretion sequence SEQ ID NO: 54), Metridia longa (Clontech), pNFkB-L27V(native secretion sequence; SEQ ID NOs: 465 and 466) or fireflyluciferase (Luc2; pGL4.32-based) plasmid DNA using FUGENE® HD (PromegaCorp.) according to the manufacturer's instructions. Cells wereincubated at 37° C., 5% CO₂ for 8 hrs, then trypsinized in 0.5 mL TrypLE(Invitrogen). The lysates were then resuspended in 8 mL DMEM with 10%FBS, 1×NEAA and 1× sodium pyruvate. 100 μL of the resuspended sample wasthen added to wells of a 96-well plate and incubated for 16 hrs at 37°C., 5% CO₂.

Following incubation, the media was removed from the cells and replacedwith 100 μL fresh media with our without TNFα (serially diluted). Toassay for secretion, at 3 and 6 hrs, 5 μL of media (in triplicate) wasremoved from the cells, brought to 50 μL with PBS and mixed with 50 μLassay reagent (as previously described with 100 μM PBI-3939).Luminescence was measured at 0 and 10 min as previously described (FIG.60).

For measuring Metridia longa luciferase activity, the Ready-To-Glow™Secreted Luciferase System (Clontech) was used according to themanufacturer's protocol. Briefly, 5 μL Ready-to-Glow™ reagent was addedto 5 μL of sample and 45 μL of PBS. Luminescence was measuredimmediately after reagent addition (FIG. 60).

E. L27V Optimized Variants.

Plasmid DNAs (pGL4.32-L27V00, pGL4.32-L27V01, pGL4.32-L27V02,pGL4.32-L27V03, and pGL4.13) were prepared for transfection usingFUGENE® HD according to the manufacturer's protocol. The pGL4.32 vector(Promega Corp.) contains the NF-κB response element. The L27V codonoptimized sequences replaced the Luc2P sequence in the vector. pGL4.13vector (Promega Corp.) contains the Luc2 gene driven by the SV40promoter.

300 μL of DNA transfection mixture was then mixed with 6 mL of HeLa cellsuspension (2×10⁵ cells/mL), homogenized, and 100 μL plated into wellsof a 96-well plate. The cells were then incubated overnight at 37° C.,5% CO₂. Following incubation, 10 μL of 10×rhTNFα in DPBS with BSA wasadded to the wells and incubated for 4.5 hrs at 37° C., 5% CO₂. Sixwells were given vehicle only. The cells were then allowed toequilibrate at room temperature for 20 min, and then 100 μL assayreagent (as previously described with 100 μM PBI-3939) was added. Tocells expressing Luc2 or receiving vehicle only treatment, 100 μL of theONE-GLO™ Luciferase Assay Reagent was added. Luminescence was measured12 min post-assay reagent addition as previously described. FIGS. 61A-Bshows the absolute luminescence, FIGS. 61C-D shows normalizedluminescence and FIGS. 61E-F shows fold response.

Example 37 OgLuc Variants in a Transcription Reporter Assay

To demonstrate the ability of the OgLuc variants of the presentinvention to be used as transcription reporters, the OgLuc variant 9B8opt was used as a transcriptional reporter in a forward, reverse, andbulk transfection. These methods of transfection were chosen becausethey are representative of the approaches commonly used for thetransient expression of genetic transcriptional reporters.

Forward Transfection

Transcriptional reporters containing the cAMP response element (CRE) and9B8 opt or 9B8 opt further comprising the PEST protein degradationsequence (9B8 opt-P) were prepared in the pGL4.29 (Promega Corp.)backbone, i.e., the luc2P gene of the pGL4.29 vector was replaced with9B9 opt (SEQ ID NO: 24) or 9B8 opt-P (SEQ ID NO: 65). pGL4.29 was usedas a control/benchmark.

HEK293 cells were plated at 15,000 cells/well in six 96-well tissueculture plates. Cells were grown in 100 μL of DMEM+10% FBS+1×non-essential amino acids (NEAA) and incubated overnight at 37° C. Thecells were transiently transfected with either 10 ng or 100 ng plasmidDNA/well of pGL4.29 9B8 opt, pGL4.29 9B8 opt-P, or pGL4.29. Plasmid DNAwas mixed with 850 μL of OPTI-MEM® (Invitrogen) and 32.4 μL of FUGENE®HD transfection reagent (Promega Corp.) and incubated at roomtemperature for 10 min. Eight μL of the transfection/reporter DNAmixture was added to the appropriate wells (2 constructs/plate). Cellswere incubated for 4 hrs at 37° C. The medium was replaced withOPTI-MEM®+0.5% dialyzed FBS+1×NEAA+1× sodium pyruvate+1× Penn-Strep andincubated overnight at 37° C.

Following incubation, 10 nM or 10 μM FSK (from a 10× stock) in OPTI-MEM®was added to the cells and incubated for 3 hrs at 37° C. A lytic reagentcontaining 100 mM MES pH 6.1, 1 mM CDTA, 150 mM KCl, 35 mM thiourea, 2mM DTT, 0.25% TERGITOL® NP-9 (v/v), 0.025% MAZU® DF 204, and 20 μMPBI-3939 was added to the cells containing pGL4.29 9B8 opt or pGL4.299B8 opt-P and allowed to incubated for 10 min at room temperature (100μL lytic reagent added to 100 μL cells). ONE-GLO™ assay reagent (PromegaCorp.) was added to cells containing pGL4.29 and used according to themanufacturer's protocol (100 μL reagent added to 100 μL cells).Luminescence was measured on a GLOMAX® Luminometer. Table 26 shows theluminescence of the HEK293 cells expressing the transcriptionalreporters containing CRE treated with 10 nM (“baseline”) or 10 mM FSK,and the response to FSK (i.e., the luminescence generated by the 10 mMFSK treated cells divided by the luminescence generated of the 10 nM FSKtreated cells.)

The results shown in Table 28 indicate that 9B8 opt and 9B8 opt-P werebrighter than luc2P, and that all the luciferase reporters responded toFSK when 100 ng of DNA was used for the transfection. However, when only10 ng of DNA was used for the transfection, the luminescence for theluc2P reporter was below the detection level for the luminometer.

TABLE 28 Transcriptional Reporters Containing CRE in HEK293 Cells (3 htimepoint) 100 ng DNA for transfection 10 ng DNA for transfectionReporter RLU RLU construct baseline (10 mM FSK) Response baseline (10 mMFSK) Response 9B8 opt 3,078,418 104,687,723 34 192810 12,926,465 67 9B8opt-P 122,071 20,544,753 168 11179 1,353,459 121 luc2P 356 5,293 15 0 0—

Reverse Transfection

Transcriptional reporters containing the antioxidant response element(ARE) and 9B8 opt or 9B8 opt-P were prepared in the pGL4.29 (PromegaCorp.) backbone, i.e., the luc2P gene of the pGL4.29 vector was replacedwith 9B9 opt or 9B8 opt-P, and CRE was replaced with 2×ARE (SEQ ID NO:66) using methods known in the arts.

HEK293 cells were trypsinized (T75 flask, 3 mL trypsin) and resuspendedin 1×10⁵ cells/mL (approximately 8.9×10⁶ total cells) in mediumcontaining DMEM+10% FBS+1×NEAA. Each transcriptional reporter wasprepared for transfection by mixing 1.2 mL OPTI-MEM®, 12 μLtranscription reporter DNA (100 ng) and 36 μL FUGENE® HD transfectionreagent together and incubated at room temperature for 35 min. Followingincubation, 624 μL of the transfection/reporter DNA mixture was added to12 mL of cell suspension and mixed by inversion. After mixing, 100 μL ofthe cell/DNA mixture was added to wells of a 96-well plate (2constructs/plate). The cells were incubated at 37° C. for 22 hrs.Tert-butylhydroquinone (a Nrf2 stabilizer; tBHQ; 1 μM (“baseline”) or 20μM) or sulphoraphane, (an organosulfer antioxidant known to activateNrf2; 1 μM (“baseline”) or 20 μM) in OPTI-MEM® was added to each welland incubated at 37° C. for 24 hrs. Cells were lysed with 100 μL lyticreagent as described above for the forward transfection. Luminescencewas measured on a GLOMAX® Luminometer.

Table 29 shows the luminescence of the HEK293 cells expressing thetranscriptional reporters containing ARE treated with 1 μM (“baseline”)or 20 μM sulphoraphane and the response to sulphoraphane (i.e., theluminescence generated by the 1 μM sulphoraphane treated cells dividedby the luminescence generated of the 20 μM sulphoraphane treated cells).Table 30 shows the luminescence of the HEK293 cells expressing thetranscriptional reporters containing ARE treated with 1 μM (“baseline”)or 20 μM tBHQ, and the response to tBHQ (i.e., the luminescencegenerated by the 1 μM tBHQ treated cells divided by the luminescencegenerated of the 20 μM tBHQ treated cells). Tables 29 and 30 show that9B8 opt and 9B8 opt-P could report the presences of two different knownstimuli for ARE.

TABLE 29 Transcriptional Reporters Containing ARE in HEK293 Cells (24 htime point) 100 ng DNA for transfection RLU (20 mM Reporter constructbaseline sulphoraphane) Response 9B8 opt 15,600,000 89,600,000 5.8 9B8opt-P 258,406 3,940,000 15

TABLE 30 Transcriptional Reporters Containing ARE in HEK293 Cells (24 htime point) 100 ng DNA for transfection Reporter construct baseline RLU(20 mM tBHQ) Response 9B8 opt 15,100,000 120,000,000 8 9B8 opt-P 317,2388,460,000 27

Bulk Transfection

The transcriptional reporters containing CRE and 9B8 opt or 9B8 opt-Pdescribed in the forward transfection were used in the bulk transfectionof HEK293 and NIH3T3 cells. Transcriptional reporters containing theheat shock response element (HRE; SEQ ID NO: 67) and 9B8 opt or 9B8opt-P were prepared in the pGL4.29 (Promega Corp.) backbone, i.e., theluc2P gene of the pGL4.29 vector was replaced with 9B9 opt or 9B8 opt-P,and the CRE was replaced with HRE. The transcriptional reportercontaining FIRE and 9B8 opt-P was used in the bulk transfection of HeLacells

HEK293, NIH3T3, or HeLa cells were plated to a single well of a 6-welltissue culture plate the day before transfection at a density of 4.5×10⁵cells/well in 3 mL complete medium (DMEM+10% FBS+1×NEAA+1× sodiumpyruvate) for HEK293 cells, 3×10⁵ cellsNvell in 3 mL complete medium(DMEM+10% fetal calf serum (FCS)+1×NEAA+1× sodium pyruvate) for NIH3T3cells, or 9.9×10⁵ cells/well in 3 mL complete medium (DMEM+10%FBS+1×NEAA) for HeLa cells. Cells were grown overnight at 37° C.

3,300 ng of reporter plasmid DNA in 155 4 OPTI-MEM® was mixed with 9.9μL FUGENE® HD transfection reagent, vortexed briefly, and incubated atroom temperature for 10 min. The CRE transcriptional reporters were usedto transfect HEK293 and NIH3T3 cells. The HRE transcriptional reporterswere used to transfect HeLa cells. The reporter mixture was added tocells and mixed by gentle rocking followed by incubation at 37° C. for 6hrs (HEK293 and NIH3T3) or 3 hrs (HeLa). Cells were then trypsinized andresuspended in medium (DMEM+10% FBS+1×NEAA+1× sodium pyruvate for HEK293cells, DMEM+10% FCS+1×NEAA+1× sodium pyruvate for NIH3T3 cells, orDMEM+10% FBS+1×NEAA for HeLa cells), followed by plating to theindividual wells of a 96-well plate (20,000 cells/100 μL for HEK293,10,000 cells/100 4 for NIH3T3, or 13,000 cells/4 for HeLa) and incubatedat 37° C. overnight.

FSK (CRE stimulator) or 17-AAG (HRE stimulator; 17-Allylamino-demethoxygeldanamycin) in OPTI-MEM® was added to the cells (10 nM or 10 μM finalconcentration for FSK; 1 nM or 1 μM final concentration for 17-AAG) andincubated at 37° C. for 4 hrs (FSK) or 6 hrs (17-AAG). Plates wereremoved from the incubator and allowed to equilibrate to roomtemperature for 25 min. Cells were lysed with 100 μL lytic reagent asdescribed above for the forward transfection. Luminescence was measuredon a GLOMAX® Luminometer.

Table 31 shows the luminescence of the HEK293 cells expressing thetranscriptional reporters containing CRE treated with 10 nM (“baseline”)or 10 mM FSK and the response to FSK. Table 32 shows the luminescence ofthe NIH3T3 cells expressing the transcriptional reporters containing CREtreated with 10 nM (“baseline”) or 10 mM FSK and the response to FSK.Table 33 shows the luminescence of the HeLa cells expressing thetranscriptional reporters containing HRE treated with 10 nM (“baseline”)or 10 mM 17-AAG and the response to 17-AAG.

Tables 29-31 show that 1) both versions of the 9B8opt OgLuc variant canreport the presence and stimulatory effects of FSK on CRE in the contextof two different cell lines, HEK293 and NIH3T3, and 2) 9B8 optP canreport the presence and stimulatory effects of 17-AAG on HRE in thecontext of HeLa cells.

TABLE 31 Transcriptional Reporters Containing CRE in HEK293 Cells (4 htime point) 100 ng DNA for transfection Reporter construct baseline RLU(10 mM FSK) Response 9B8 opt 39,700,000 654,000,000 16 9B8 opt-P3,960,000 460,000,000 116

TABLE 32 Transcriptional Reporters Containing CRE in NIH3T3 Cells (4 htime point) 100 ng DNA for transfection Reporter construct baseline RLU(10 mM FSK) Response 9B8 opt 9,187,000 23,600,000 2.6 9B8 opt-P 410,4613,720,000 9

TABLE 33 Transcriptional Reporters Containing HRE in HeLa Cells (6 htime point) 100 ng DNA for transfection Reporter construct baseline RLU(1 mM 17-AAG) Response 9B8 opt-P 278,118 3,204,000 12

Example 38 Lytic and Secretable Reporter in Difficult to Express Cells

HepG2 cells, 1×10⁵ cells/mL in a cell suspension, were reversetransfected with plasmid DNA (pGL4.32 backbone; Promega Corp.)containing L27V02, luc2P (Promega Corp.), luc2 (Promega Corp.) orL27V02-IL6 (L27V02 with the native secretion sequence replaced with theIL-6 secretion sequence; (“IL601-L27V02A”; SEQ ID NO: 324) using FUGENE®HD according to the manufacturer's instructions (1:20 DNA-transfectionmixture to cells). 100 μL cell suspension was then plated into wells ofa 96-well plate and incubated for 22 hrs at 37° C., 5% CO₂. Other OgLucconstructs which have the native secretion sequence replaced by the IL-6secretion sequence include IL601-L27V01 and IL602-L27V03 (SEQ ID NOs:321 and 327, respectively).

For secretion analysis, the media was removed from the cells, and thecells washed in 100 μL DPBS. 100 μL complete media (DMEM+10% FBS+1×NEAA)was added along with varying doses (1 pg/mL-100 ng/mL) of rhTNFα(“TNFα”) for 4.5 h. 10 μL of the media was then removed, added to 90 μLcomplete media, and 100 μL assay reagent (as previously described; 100μM PBI-3939) added. Luminescence was measured as previously described(FIG. 62A).

For lytic analysis, following plating, the cells were incubated for 4.5hrs at 37° C., 5% CO₂. The cells were then allowed to equilibrate toroom temperature for 20 min. Assay reagent (as previously described; 100μM PBI-3939) was added to the cells, and luminescence measured aspreviously described (FIG. 62B).

Example 39 Additional Lytic Reporter Features

The OgLuc variants of the present invention in the context of acell-based, lytic transcriptional reporter should offer a luminescentsignal of a magnitude such that the signal appears sooner than it mightwith other luciferases. The bright luminescence should also allow forweak promoters to be examined.

Example 40 Mammalian Cell Transfections

The OgLuc variants of the present invention were used as reporters indifficult to transfect cell lines, e.g., Jurkat, HepG2, primary cells,non-dividing primary cells, or stem cells. (See e.g., FIG. 59C) Due totheir high signal intensity, the OgLuc variants enable detectableluminescence when transfection efficiency is low. The OgLuc variants canalso be used as reporters in cells that are especially sensitive toconditions associated with transfection, i.e., DNA concentration,transfection reagent addition. Due to the brightness of the OgLucvariants, an adequate level of luminescence can be achieved using lowerDNA concentrations, less transfection reagent, and perhaps shorterpost-transfection times prior to beginning an assay. This will placeless of a toxicity burden on what would otherwise be sensitive cells.The bright luminescence of the OgLuc variants should also allow for asignal to be detected at very long time points in the event such anoutput is desirable. As another example, the OgLuc variants could beused as reporters for single copy native promoters, e.g., HSBthymidylate kinase (TK) promoter, HOX genes, or LIN28.

Example 41 Stable Cell Lines

The identification of robust, stable cell lines expressing an OgLucvariant of the present invention, either in the cytoplasm or as asecreted form, can be facilitated by the bright signal of the luciferaseand the small size of the OgLuc gene. The relatively small gene sequenceshould reduce the likelihood of genetic instability resulting from theintegration of the foreign DNA.

To generate stable cell lines using an OgLuc variant of the presentinvention, plasmid DNA comprising a nucleotide sequence for an OgLucvariant and a selectable marker gene, e.g., neomycin, hygromycin, orpuromycin, is used to transfect a cell line of interest, e.g., HEK293cells. Cells of an early passage number, e.g., less than 10 passages,are plated into T25 (1×10⁶) or T75 (3×10⁶) tissue culture flasks andallowed to grow overnight to approximately 75% confluency. Cells arethen transfected using the above plasmid DNA and an appropriatetransfection reagent, e.g., TRANSIT0-LT1 or FUGENE® HD. Forty-eight hrspost-transfection, the media is replaced on the cells with selectionmedia containing the selection drug, e.g., G418, hygromycin orpuromycin, at a concentration previously determined to killuntransfected cells. Selection of cells containing the plasmid DNAoccurs over 2-4 weeks. During this time, the cells are re-plated inselection media at various concentrations into either T25 or T75 tissueculture flasks. The media on the re-plated cells is replaced every 3-4days for 2-3 weeks with fresh selection media. The flasks are monitoredfor the formation of live cell colonies. Eventually, the flasks willcontain many large colonies and few dead cells.

From the pool of stable colonies in the flasks, single colonies areisolated and expanded into a single 24-well tissue culture plate.Briefly, cells are harvested using the trypsin/EDTA method, i.e., cellsare harvested by removing media, rinsing with Ca²⁺ and Mg²⁺ free PBS anddetached by treatment with Trypsin/EDTA. The cells are counted using ahemocytometer and diluted 1×10⁵ in complete media. The cells are thendiluted to 100 cells/mL, 33 cells/mL, 10 cells/mL, and 3.3 cells/mL incomplete media. 100 μL of each dilution is plated into all wells of96-well tissue culture plate (1 plate for each dilution) and allowed togrow 4-5 days after which 50 μL of selection media is added to thecells. Approximately a week after plating, cells are visually screenedfor colony growth and another 50 μL of selection media is added. Thecells continue to be monitored until a single colony covers 40-60% ofthe well area. When a colony is ready for expansion and screening,colonies are harvested using the trypsin/EDTA method. Each colony istransferred to selection media as follows: 1) Dilute 1:10 into 6 wellsof a 96-well assay plate for functional assay, e.g., luminescencedetection; 2) Dilute 1:10 into 3 wells of a clear bottom 96-well assayplate for cell viability assay, e.g., CELLTITER-GLO® Luminescent CellViability Assay (Promega Corp.); and 3) Dilute 1:10 into a 24-welltissue culture plate for expansion. Cells in the plates for thefunctional and cell viability assay are then grown 2-3 days and thefunctional and cell viability assays performed. Positive clones in the24-well plate are further tested with the functional and cell viabilityassays as well as for stability of expression and response for at least20 passages, normal growth rate morphology, and frozen for future use atthe earliest possible passage.

Example 42 OgLuc Secretion Signal Analysis

A. IV opt

The wild-type OgLuc is processed after synthesis into a mature proteinwith the secretion signal sequence cleaved off. To determine if thesecretion signal sequence would facilitate secretion of the OgLucvariant, the IV opt variant of Example 25 and hRL were cloned into pF4Agcontaining an N-terminal OgLuc secretion signal (SEQ ID NO: 54). HEK293cells (15,000) in 100 μL Dulbecco's Modified Eagle's medium (“DMEM”)with 10% fetal bovine serum (FBS) were transfected as described inExample 25 with 100 ng of plasmid DNA, i.e., hRL or IV opt with orwithout the secretion signal and grown overnight at 37° C. 50 μL ofmedia was removed to a new plate and saved for a later assay generatinga “media” sample. The rest of the media was removed, and the cells werelysed with 100 μL of lysis buffer described in Example 25 to generate a“lysate” sample. 10 μL of media sample and 10 μL of lysate sample wereassayed for luminescence (FIG. 63). Samples for hRL with (“Renilla sig”)or without (“Renilla”) the OgLuc secretion signal sequence were measuredusing 50 μL of lysis buffer containing 20 μM native coelenterazine.Samples for IV opt with (“IV opt sig”) or without (“IV opt”) the OgLucsecretion signal sequence were measured using 50 μL of lysis buffercontaining 20 μM PBI-3939.

In FIG. 63, the filled bars represent the amount of light that wasdetected from the media in the absence of any lytic reagent. The openbars represent the total light (secreted+non-secreted) that was detectedupon addition of a lytic reagent. FIG. 63 shows that N opt was secretedfrom HEK293 cells into the growth media and that the secretion signalsequence was functional in mammalian cells. “IV opt sig” represents theonly situation where a significant amount of luciferase was detected inthe media. The results also indicate that this particular signal peptidedid not facilitate secretion of hRL.

B. 9B8, V2 and L27V

To determine if the secretion signal sequence of OgLuc facilitates itssecretion, the OgLuc variants 9B8, V2 and L27V were cloned into pF4Agcontaining an N-terminal OgLuc secretion signal sequence. The variantswere also cloned into vectors without the secretion signal sequence. CHOor HeLa cells were then plates at 100,000 cells/well in 1 mL F12 mediawith 10% FBS and 1× sodium pyruvate (CHO cells) or DMEM with 10% FBS and1× sodium pyruvate (HeLa cells) into 12-well plates and incubatedovernight at 37° C., 5% CO₂.

After the overnight incubation, the cells were transfected with 1 μgplasmid DNA containing 9B8, V2, or L27V with or without the secretionsignal sequence using the TRANSIT®-LT1 transfection reagent (Minis Bio)and OPTI-MEM® media (Invitrogen). The cells were again incubatedovernight at 37° C., 5% CO₂.

After the second overnight incubation, the media was removed and savedfor analysis. To the cells, 1 mL of assay buffer (1 mM CDTA, 150 mM KCl,2 mM DTT, 100 mM MES pH 6.0, 35 mM Thiourea and 0.5% TERGITOL® NP-9(v/v)) was added to create a cell lysate. To 10 μL of cell lysate orsaved media from each sample, 50 μL assay buffer with 40 μM FBI-3939 wasadded, and luminescence measured as described above. FIGS. 64A-Ddemonstrates that 9B8, V2 and L27V variants can be used in a secretablesystem.

To determine the stability of the secreted variants, 150 μL aliquots ofthe saved media from each sample was placed at 37° C. or 50° C. Thealiquots were then removed at different time points (0, 1, 2, 3, 5, 6,and 7 min), frozen on thy ice, and kept at −20° C. until assayed. Toassay for stability, the media aliquots were thawed to room temperature,and 10 μL of each aliquot was mixed with assay buffer with PBI-3939 (pH6.0) as described above. Luminescence was measured as above, and thehalf-life (t₅₀) determined (Table 34).

TABLE 34 sample ½ life 37° C. (days) 9B8 8 V2 10 L27V 17 sample ½ life50° C. (hours) 9B8 3 V2 7 L27V 11

C. 9B8 and V2 Comparison to Secreted Luciferase of Metridia longa

The secretion of the OgLuc variants 9B8 and V2 was compared to that ofthe secreted luciferase from Metridia longa. CHO cells were plated at300,000 cells/well in 3 mL F12 media with 10% FBS into wells of 6-wellplates and incubated overnight at 37° C., 5% CO₂. The cells were thentransfected with either 10 or 100 ng of each variant or Metridialuciferase (Clontech) plasmid DNA using TRANSIT®-LTI according to themanufacturer's instructions and incubated for 20 hrs at 37° C., 5% CO₂.After transfection, the media was removed from the cells and assayed.For the OgLuc variants, 50 μL of media was assayed with 50 μL of assayreagent (previously described; 40 μM PBI-3939). For Metridia luciferase,the media was assayed using the Ready-to-Glo™ Secreted LuciferaseReporter System (Clontech) according to the manufacture's protocol.Briefly, 5 μL of the 1× substrate/reaction buffer was added to 50 μL ofmedia sample. Luminescence was then measured as previously described(FIGS. 65A-B).

Example 43 Evaluation of OgLuc Variants and Novel Coelenterazine in LiveCells

A. The use of OgLuc variants and PBI-3939 in live cells was examined.HEK293 cells were plated in 96-well plates at 15,000 cells/well andgrown overnight, at 37° C. The following day, the cells were transientlytransfected using TRANSIT®-LT1 in 3 replicates with 100 ng of hRL or 9B8opt in pF4Ag and grown overnight at 37° C. The following day the growthmedia was removed and replaced with media containing 60 μM VIVIREN™ LiveCell Substrate (Promega Corp.), 60 μM ENDUREN™ Live Cell Substrate(Promega Corp.), or 60 μM PBI-3939 for both hRL and 9B8 opt transfectedcells. Non-transfected cells were used as background control. The platewas incubated at 37° C. during the course of one day and periodicallymeasured on a TECAN® GENIOS™ Pro luminometer, i.e., 11 times over thecourse of 24 hrs. FIGS. 66A-B shows the luminescence of the transfectedcells divided by the luminescence of the non-transfected cells for eachof the substrates, i.e., the signal to background ratio. The data showsthat 9B8 opt generated luminescence in a live cell setting (i.e., nolysis) by incubating cells with VIVIREN™, ENDUREN™, or PBI-3939. Thedata also demonstrated that PBI-3939 can permeate cells in culture,react with the OgLuc variant, and generate luminescence, thus making itcompatible with use in a live cell assay.

B. To demonstrate live cell analysis using the OgLuc variants, L27V wasfused to HALOTAG® and expressed and monitored in live cells. U20S cellswere plated at 40,000 cells/mL into imaging chamber wells and incubatedovernight at 37° C., 5% CO₂. Cells were than transfected using FUGENE®HD according to the manufacturer's protocol with the plasmids pFC14K,pFN21K or pF4Ag (all Promega Corp.) containing L27V or pF4Ag containingL27V with the native or IL-6 secretion sequence. Cells were thenincubated for 24 hrs at 37° C., 5% CO₂.

Following incubation, the cells were exposed to HALOTAG® TMR ligand(Promega Corp.), imaged, and fixed. Immunocytochemistry (ICC) was thenperformed according to the ICC protocol in the HALOTAG® Technology:Focus on Imaging technical manual (Promega Corp.; TM260). The primaryantibody used was a polyclonal rabbit, anti-OgLuc 9B8 antibody (1:1000).The secondary antibody used was an Alexa 488 conjugated secondaryantibody (green) (FIG. 67A). FIG. 67A shows green fluorescent channeland FIG. 67B shows the differential interference contrast (DIC). Imageswere acquired using an Olympus Fluoview FV500 confocal microscope(Olympus, USA) outfitted with a 37° C.+CO2 environmental chamber (SolentScientific Ltd., UK).

FIGS. 67B-D shows the ICC images with native or IL-6 secretion sequence.Both signal sequences dramatically decrease the amount of enzyme in thenucleus. The punctuate nature of the labeling in the cytoplasm isindicative of vesicle formation expected to occur during the secretionprocess. The data demonstrates that the presence of a signal peptidereduces the amount of luciferase in the nucleus.

C. As shown above, the OgLuc variants and novel substrates of thepresent invention are biocompatible. A reporter system is envisionedwhere the OgLuc variant is cloned into an expression vector with apromoter of interest and expressed in cells as a reporter protein. Thecells are then treated with PBI-3939 which will permeate cells inculture, react with the OgLuc variant, and generate luminescence.

In addition to being cell permeant, PBI-3939 shows comparablebiocompatibility to native coelenterazine in terms of cell viability. Aversion of compound 3939 containing chemical modifications known toincrease the stability of native coelenterazine in media can besynthesized and used for more robust, live cell OgLuc variant-basedreporter assays. Another example of live cell reporting includes the useof a secretable OgLuc variant as a reporter. The native secretion signalpeptide (or other known secretion signal peptides) can be fused to theN-terminus of an OgLuc variant so that when the fusion is expressed inmammalian cells, a portion of it will be secreted through the cellmembrane into the culture media. Upon addition of substrate luminescenceis generated.

Example 44 Protein Fusion Reporters

The OgLuc variants of the present invention can be used as fusion tagsfor a target protein of interest as a way to monitor intracellularlevels of that target protein. Specific proteins involved in stressresponse pathways, e.g., DNA damage, oxidative stress, inflammation, canbe monitored in cells as a way to probe the role various types ofstimuli may play in these pathways. The variants can also be used as ameans to monitor cellular trafficking of a target protein. The variantscan also be fused to viral genomes (e.g., HIV, HCV) so that titerlevels, i.e., infectivity, can be monitored in cells following treatmentwith potential antiviral agents. The variants can also be fused to greenfluorescent protein (GFP) or HALOTAG® (in addition to a target protein)so that FACS could be used to identify high expression clones and toprovide localization information.

Example 45 Evaluation of OgLuc Variant in 3-Component Fusion Protein(“Sandwich”)

3-component fusion proteins, or “sandwich” fusions, can be used to placebioluminescent and fluorescent proteins close to one another foroptimization of a biosensor based on BRET.

A. C1+4AE, IV, 9B8 and 9F6

The OgLuc variants C1+4AE (SEQ ID NOs: 55 and 56), IV (SEQ ID NOs: 57and 58), 9B8 (SEQ ID NOs: 61 and 62), and 9F6 (SEQ ID NOs: 63 and 64),and hRL (SEQ ID NOs: 32 and 33) were cloned into a pF4Ag fusion vectorwith an N-terminal Id (Benezra et al., Cell, 61(1):49-59 (1990)), knownto be a poor fusion partner, and a C-terminal HT7, which was used fornormalization. The gene of interest was “sandwiched” between Id and HT7,i.e., Id-Luciferase-HT7. E. coli lysates, containing the variantconstructs in pF4Ag or pF4Ag sandwich background, were prepared asdescribed in Example 26 and then assayed with 20 μM nativecoelenterazine in the buffer described in Example 25.

FIG. 68 shows the luminescence for each variant in either pF4Ag or pF4Agsandwich background (“Sand”). FIG. 69 shows the fold-decrease inluminescence due to the presence of Id and HT7 and determined bydividing the luminescence of the variant in pF4Ag by the luminescence ofthe variant in the pF4Ag-sandwich. Samples with the largest valuesshowed the most sensitivity to the poor fusion partner Id. The variant9B8 was the brightest in the sandwich context.

B. 9B8 OPT and 9B80PT-FIC33N

The variants 9B8 opt and 9B8 opt+K33N were analyzed in a sandwichbackground as described above. Sandwich constructs for 9B8 opt (SEQ IDNOs: 40 and 41) and 9B8 opt+K33N (SEQ ID NOs: 59 and 60) were generatedas described above. E. coli lysates were assayed and measured using thesame assay buffer and luminometer as used for generating FIG. 40. FIG.70 shows the fold-decrease in the presence of a sandwich backgroundindicating that 9B8 opt+K33N is less sensitive to the poor fusionpartner Id than 9B8 opt.

C. 23D2 and 24C2

Variants 23D4 (NF) and 24C2 (NF) were subcloned into the Id-OgLuc-HT7sandwich background and assayed in E. coli. The sandwich variants, 23D4(F) (SEQ ID NOs: 76 and 77) and 24C2 (F) (SEQ ID NOs: 78 and 79) werecompared to 9B8 opt+K33N in the sandwich background (SEQ ID NO: 59 and60). Table 35 shows the variants had at least the same luminescence as9B8 opt+K33N in the sandwich background context.

TABLE 35 Increase in Luminescence Generated by OgLuc Variants Comparedto 9B8 opt + K33N + 170G in Sandwich Background Fold over 9B8 opt +Sample Sequence K33N sandwich (E. coli) 23D4 (F) G26G, M106L, R112R,170G 1.0 24C2 (F) R11Q, T39T, 170G 1.0

D. 1F7 and 15H1

The PCR library in the Id-OgLuc-HT7 sandwich background was screened foradditional variants with increased luminescence compared to 9B8 opt+K33Nin sandwich background. Selected variants were then assayed in HEK293and NIH3T3 cells. Table 36 shows the fold-increase in luminescence ofthe sandwich variants in E. coli, HEK293 and NIH3T3 cells, and the aminoacid substitutions found in the variants. 1F7 (F) (SEQ ID NOs: 84 and85) and 15H1 (F) (SEQ ID NOs: 86 and 87) had at least 1.3 fold-increasein luminescence in E. coli. 1F7 (F) was brighter than 9B8 opt+K33N inthe sandwich background in HEK293 and NIH3T3 cells.

TABLE 36 Increase in Luminescence Generated by OgLuc Variants Comparedto 9B8 opt + K33N in Sandwich Background Fold over 9B8 opt + K33Nsandwich Sample Sequence E. coli HEK293 NIH3T3 1F7 (F) K43R, Y68D 1.92.4 1.4 15H1 (F) D19D, S66N 1.5 0.9 1.2

The sandwich variants were subcloned into the pF4Ag-based non-fusionbackground vector to generate 1F7 (NF) (SEQ ID NOs: 80 and 81) and 15H1(NF) (SEQ ID NOs: 82 and 83) and were analyzed as described above andcompared to 9B8 opt+K33N. Table 37 shows the fold-increase inluminescence of the variants in E. coli, HEK293 and NIH3T3 cells. 1F7(NF) and 15H1 (F) had at least 1.3 fold-increase in luminescence in E.coli and HEK293 cells.

TABLE 37 Increase in Luminescence Generated by OgLuc Variants Comparedto 9B8 opt + K33N + 170G Fold over 9B8 opt + K33N + 170G Sample SequenceE. coli HEK293 NIH3T3 1F7 (NF) K43R, Y68D 1.5 1.5 1.1 15H1 (NF) D19D,S66N 1.7 1.7 1.2

E. V2, 9B8 opt+K33N+L27V+K43R+Y68D, 9B8 opt+K33N+L27V+T39T+K43R+S66N andL27V

The variants 9B8 opt+K33N+T39T+K43R+Y68D (“V2”; SEQ ID NOs: 92 and 93),9B8 opt+K33N+L27V+K43R+Y68D (SEQ ID NOs: 339 and 340), 9B8opt+K33N+L27V+T39T+K43R+S66N (SEQ ID NOs: 341 and 342), and 9B8opt+K33N+L27V+T39T+K43R+Y68D (“L27V”; SEQ ID NOs: 88 and 89) weresubcloned into the Id-OgLuc-HT7 sandwich background as described aboveand assayed in HEK293 and NIH3T3 cells as described above. Theluminescence generated by the sandwiched variants were compared to theluminescence generated by the 9B9 opt+K33N sandwich (SEQ ID NOs: 59 and60) (Table 38). The L27V sandwich (SEQ ID NOs: 90 and 91) and V2sandwich (SEQ ID NOs: 94 and 95) had at least 1.3× fold-increase inluminescence in HEK293 and NIH3T3 cells.

TABLE 38 Increase in Luminescence Generated by OgLuc variants insandwich background compared to 9B8 opt + K33N in sandwich backgroundSample NIH 3T3 cells HEK 293 K33N Sand 1.0 1.0 T39T, K43R, Y68D Sand 1.62.3 L27V, K43R, Y68D Sand 1.4 1.7 L27V, T39T, K43R, S66N Sand 0.7 0.7L27V, T39T, K43R, Y68D Sand 1.4 1.7

The sandwich and non-sandwich versions of the variants V2, 9B8opt+K33N+L27V+K43R+Y68D, 9B8 opt+K33N+L27V+T39T+K43R+S66N, and L27V wereassayed in HEK293 and NIH3T3 cells as described in Example 37. Theluminescence generated by the non-sandwiched variants was compared tothe luminescence generated by the sandwiched variants (Table 39). Thedata shown in Table 39 indicates that the fold-decrease in luminescencefor the 9B8 opt+K33N sandwich was less in mammalian cells than in E.coli cells as shown in FIG. 70.

TABLE 39 Fold-Decrease in Luminescence of the OgLuc Variants in thePresence of Sandwich Background Sample NIH 3T3 cells HEK 293 K33N 29 15T39T, K43R, Y68D 20 6 L27V, K43R, Y68D 22 8 L27V, T39T, K43R, S66N 25 12L27V, T39T, K43R, Y68D 18 6

Example 46 Multiplexing

A. Lysates of E. coli expressing the variant 9B8 opt were prepared aspreviously described in Example 27 and diluted 1000-fold in DMEM withoutphenol red+0.1% PRIONEX®. Luminescence from a sample containing 6.3μg/mL of purified red click beetle luciferase and E. coli lysateexpressing the variant 9B8 opt was detected using a modified DUAL-GLO®Luciferase Assay System (Promega Corp.). DUAL-GLO® STOP&GLO® Reagentcontaining 20 μM coelenterazine-h and DUAL-GLO® STOP&GLO® Reagentcontaining 20 μM PBI 3939 were used, according to the manufacturer'sprotocol, to detect the red click beetle luciferase and OgLuc variant9B8 luciferase from a single sample. Three replicates were performed.

Luminescence was detected on a Turner MODULUS™ luminometer. Table 40shows the average luminescence generated by the red click beetleluciferase (“click beetle”), and the luminescence generated by 9B8 opt(“Ogluc”) with coelenterazine-h (“coel h”) or PBI-3939 (“3939”). Thestandard deviation (“+/−”) and coefficient of variance (“CV”) are alsoshown. A “no coelenterazine” control was performed to illustrate theamount of quenching of the red click beetle signal by the DUAL-GLO®STOP&GLO® Reagent of the DUAL-GLO® Luciferase Assay System in theabsence of coelenterazine. The “no coelenterazine” control yielded a349-fold quench. Table 40 shows that large luminescent signals from boththe red click beetle and OgLuc variant 9B8 was detected in a singlesample. This demonstrates that each signal can be read sequentially in atwo-step assay, and the signal from the first enzyme can be quenchedenough to not contribute significantly to the signal from the secondenzyme.

TABLE 40 Average Luminescence Generated by the Red Click Beetle and 9B8opt Luciferases Using a Modified DUAL-LUCIFERASE ™ Reporter Assay clickbeetle +/− CV Ogluc +/− CV fold-quench +/− coel no coel 5,061,163147,145 2.9% 14,504 214 1.5% 349 coel h 5,082,100 152,254 3.0% 921,44047,623 5.2% 64 3939 5,078,547 41,753 0.8% 2,996,940 187,300 6.2% 207

B. To demonstrate that the multiplex reporter assay described abovecould be done in reverse, i.e., OgLuc luminescence detected first,quenched and a second luminescence detected, e.g., red click beetle orfirefly luciferase, various Renilla luciferase inhibitors (see U.S.Published Application No. 2008/0248511) were screened for their abilityto also inhibit OgLuc. Two different, previously identified, Renillainhibitors, PBI-3077 and 1424, were added at various concentrations (seeTable 41) to E. coli lysate samples expressing the variant 9B8 (dilutedas above) and a buffer containing 100 mM MES pH 6.0, 1 mM CDTA, 150 mMKCl, 35 mM Thiourea, 2 mM DTT, 0.25% TERGITOL® NP-9 (v/v), 0.025% MAZU®DF 204, and 20 μM PBI-3939. Luminescence was measured as describedpreviously except luminescence was measured using the GLOMAX®-MultiMicroplate luminometer (Promega Corp.; also known as Turner MODULUS™).As shown in Table 41, both compounds were able to inhibit OgLucluminescence. This demonstrates that an OgLuc variant can be multiplexedin a reporter assay with another luciferase wherein luminescence from anOgLuc variant is detected first in the reporter assay.

TABLE 41 The effect of PBI-3077 and PBI-1424 on Luminescence Generatedby Bacterial Lysates Expressing 9B8 opt Using PBI-3939 as a Substrate(mM or %) RLU +/− % control 27,794,600 626,862  100% Al (3077) 315,473,100 209,567   56% mM 0.3 22,210,433 102,888   80% 0.03 22,484,933927,459   81% AC (1424) 0.4 176,868 9,579 0.64% % 0.04 24,267,533363,861   87% 0.004 25,126,900 1,569,453   90%

C. The spectral resolution between OgLuc Variant L27V and fireflyluciferase (Fluc) was analyzed. Purified L27V (previously described;9.54 μM) in DMEM without phenol red+0.1% PRIONEX® was mixed with assayreagent (previously described) containing 20 μM PBI-3939. Purifiedfirefly luciferase enzyme (QUANTILUM® Recombinant Luciferase; PromegaCorp.; 271 ng/mL) in the same media was mixed with a test reagent (100mM HEPES, pH 7.4, 1 mM CDTA, 16 mM MgSO4, 1% TERGITOL® NP-9 (v/v), 0.1%MAZU® DF 204, 5 mM ATP, 50 mM DTI', 333 μM luciferin). Purified Renillaluciferase (5 ng/mL GST-Renilla) in 1× Renilla Luciferase Assay LysisBuffer (Promega Corp.) was mixed with 10.5 μM native coelenterazine inRenilla Luciferase Assay Buffer. Luminescence was measured after 3 minfor L27V and Fluc and after 10 min for Renilla luciferase (FIG. 71)

D. As another example, an OgLuc variant of the present invention couldbe used as transcriptional reporter and paired with either aequorin or acAMP circularly permuted firefly luciferase biosensor (or bothsimultaneously) to detect multiple pathways in a single sample, e.g.,aequorin for the detection and/or measurement of calcium, the biosensorfor the detection and/or measurement of cAMP, and an OgLuc variant formonitoring of downstream gene expression.

E. Other examples for multiplexing with the OgLuc variants of thepresent invention include:

i) Transfecting cells with constructs containing an OgLuc variant of thepresent invention and Firefly luciferase. After transfection, a firstreagent could be added to lyse the cells as well as provide thesubstrate to generate luminescence for the first luciferase.Luminescence from the first luciferase would then be measured. A secondreagent would then be added to quench luminescence from the firstluciferase as well as provide the substrate to generate luminescencefrom the second luciferase. Luminescence from the second luciferasewould then be measured. The choice of which luciferase to measure firstwould only depend on the ability to quench the luminescence from thefirst luciferase with the second reagent. For this example, luminescencefrom the OgLuc variant could be measured first as high concentrations ofluciferin (substrate for firefly luciferase) has been shown to inhibitOgLuc variant activity.

ii) Transfecting cells with constructs containing an OgLuc variant ofthe present invention and Firefly luciferase. After transfection, afirst reagent could be added which contained a live cell substrate togenerate luminescence for the first luciferase. Luminescence from thefirst luciferase would then be measured. A second reagent would then beadded to lyse the cells, quench luminescence from the first luciferaseand provide the substrate to generate luminescence from the secondluciferase. Luminescence from the second luciferase would then bemeasured. This is similar to i) except cell lysis will further limitusage of the live cell substrate and contribute to the quenching ofluminescence from the first luciferase.

iii) Transfecting cells with constructs containing an OgLuc variant ofthe present invention and Firefly luciferase. After transfection, onereagent could be added which contained substrates to generateluminescence from both luciferases, but the luminescence from eachluciferase is spectrally different. The emission max of the OgLucvariants is approximately 460 nm, and certain substrates for Fireflyluciferase, for example 5′-chloroluciferin and 5′-methylluciferin, canyield an emission max of approximately 610 nm. Therefore, although theremay be some overlap from the blue emission into the red emission, therewould be no overlap of the red emission into the blue emissionsuggesting that little to no mathematical correction would be involved.

iv) Transfecting cells with constructs containing an OgLuc variant ofthe present invention and Firefly luciferase. After transfection, onereagent could be added which contained live cell substrates to generateluminescence from both luciferases. The unique feature of this exampleis that firefly luminescence tends to shift to red at live cell assaytemperatures, e.g., 37° C., therefore, a number of different luciferinderivatives could be chosen as a live cell substrate for fireflyluciferase to generate luminescence which is spectrally different fromthat of the OgLuc variant.

v) Transfecting cells with constructs containing an OgLuc variant of thepresent invention and Renilla luciferase. After transfection, a firstreagent could be added to lyse the cells as well as provide thesubstrate to generate luminescence for the first luciferase.Luminescence from the first luciferase would then be measured. A secondreagent would then be added to quench luminescence from the firstluciferase as well as provide the substrate to generate luminescencefrom the second luciferase. Luminescence from the second luciferasewould then be measured. The choice of which luciferase to measure firstwould only depend on the ability to quench the luminescence from thefirst luciferase with the second reagent. For this example, inhibitorsto quench either the OgLuc variant or Renilla luciferase luminescencewould need to be used.

vi) Transfecting cells with constructs containing an OgLuc variant ofthe present invention and click beetle luciferase. After transfection,one reagent could be added which contained substrates to generateluminescence from both luciferases, but the luminescence from eachluciferase is spectrally different as click beetle luciferase generatesred-shifted luminescence with native luciferin.

Example 47 Circular Permutation

Two circularly permuted (CP) versions of the L27V variant were made:CP84 and CP95. The number designation refers to the N-terminal residue(e.g., “84” indicates the new N-terminus of the CP version).

To create the circular permutations, the prior N- and C-termini arefused together with no linker (“CP84 no linker” (SEQ ID NOs: 97 and 98)and “CP95 no linker” (SEQ ID NOs: 105 and 106)) or a 5 (“CP84 5AAlinker” (SEQ ID NOs: 99 and 100) and “CP95 5AA linker” (SEQ ID NOs: 107and 108), 10 (“CP84 10AA linker” (SEQ ID NOs: 101 and 102) and “CP9510AA linker” (SEQ ID NOs: 109 and 110), or 20 (“CP84 20AA linker” (SEQID NOs: 103 and 104) and “CP95 20AA linker” (SEQ ID NOs: 111 and 112)amino acid linker, (GSSGG)n (SEQ ID NO: 113) in between the N- andC-termini ends. (Note: L27V starts with the phenylalanine at theN-terminus, i.e., MVF. The “MV” is present in the “no linker” construct,but not in the “linker” constructs). Once circularly permuted, the CPL27V variants were cloned into the pF1K vector. E. coli cells weretransformed with the CP vectors and grown in minimal media using thestandard walk away induction protocol previously described. For each CPconstruct, cells were grown in 8 wells of a 96-well plate. Afterinduction, the 8 wells from each sample were pooled, and 10 μL lysed in40 μL lysis buffer (100 mM MES pH 6.0, 0.3×PLB, 0.3 mg/mL lysozyme,0.003 U/μL DNase I, and 0.25% TERGITOL® NP-9 (v/v)). The lysates werethen diluted 1:100 (CP versions with linker) or 1:1000 (non-CP versions)in lysis buffer. The CP version without linker was not diluted. Thelysates or lysate dilutions were assayed in triplicate in 50 μL assayreagent (previously described). Luminescence was measured as previouslydescribed (FIG. 72).

Example 48 Identifying Additional Sites for Circular Permutation

To identify additional CP sites, determine the impact of the CP sites onluciferase activity and investigate the use of a “tether” betweenfragments, CP constructs were made with a circular permutation made atapproximately every 3^(rd) site (i.e., amino acid) of the L27V variant(See FIG. 73E). One skilled in the art would understand that othersites, e.g., the 1^(st) and 2^(nd) site, could also be tested and usedin circular permuted OgLuc variants described herein using the methodsdescribed here. For example, the L27V variant has been found to beparticularly tolerant to circular permutation, particularly situationsin which a relatively large binding domain is placed in between thepermuted fragments (e.g. cAMP/RIIbB-based sensors). At each site, thelinker GSSGG-GSSGG-EPTT-ENLYFQS-DN-GSSGG-GSSGG (SEQ ID NO: 328) wasadded. The underscored sequence refers to a TEV protease recognitionsite. The purpose of the linker is to provide a long enough tetherbetween the two variant fragments so they can associate in a way thatproduces a functional luciferase enzyme. The TEV protease recognitionsite was used to provide a means to disrupt the tether (in the presenceof TEV protease) so that its importance to maintaining activity could beinvestigated. The use of the TEV protease recognition site created amode to predict which CP sites would be useful for proteincomplementation assays (PCA) or for biosensor applications (e.g.,insertion of a response element between the CP sites).

The activity that is seen prior to TEV cleavage represents how the twohalves of the variant enzyme behaves in a tethered state. TEV binding tothe recognition site causes cleavage, thereby separating the two halvesof the variant enzyme. Samples that have been cleaved with TEV wouldrepresent the un-induced state and provide an indication of how muchbackground could be expected. Lower activity after TEV cleavageindicates that the two halves cannot come together without induction.Samples that show a large loss in activity after TEV cleavage indicatesites that would function in PCA and biosensor applications. In the caseof PCA, the two halves of the variant enzyme would be fused to bindingpartners that are able to come together (tether) after an inducedbinding event. In the case of a biosensor, the two halves would “tether”after a binding-induced conformational change occurs. One example forPCA would be to fuse one half of L27V to FRB and the other half to FKBP.The two halves would be brought into proximity with exposure torapamycin (Banaszynski et al., J. Am. Chem. Soc, 127(13):4715-4721(2005)). One example of a biosensor application would be to insert aCyclic AMP binding domain (e.g., RIIbB) between the CP sites and inducea conformational change by binding of Cyclic AMP to the binding domain.

Once each CP L27V construct was made, the CP enzyme was expressed inwheat germ, E. coli and mammalian cells and digested with TEV proteaseto investigate luciferase activity.

1. For analysis in wheat germ, the CP constructs were expressed usingthe TnT® T7 Coupled Wheat Germ Extract System (Promega Corp.) accordingto the manufacture's instructions. The TnT® reactions were then diluted1:100 in 1×PBS+0.1% gelatin, and 20 added to 25 μL of TEV reactionreagent (5 μL 20× ProTEV buffer (Promega Corp.), 1 μL 100 mM DTT, and 2μL 10 U ProTEV Plus (Promega Corp.)). The volume of the digestionreactions was the brought to 100 μL with water and incubated at 30° C.for 60 min. Control samples without TEV protease were also prepared. 10μL of the digested samples were then added to 40 μl DMEM to a finalvolume of 50 μL and assayed in 50 μL assay reagent (as previouslydescribed; 100 μM PBI-3939). Luminescence was measured as previouslydescribed (FIGS. 73A-D).

2. For analysis in mammalian cells, HEK293 cells were transfected withthe CP L27V variants using a reverse transfection protocol. Briefly, 1ng CP L27V plasmid DNA was mixed with 1 μg carrier DNA and added tocells in a well of a 12-well plate. Cells were then incubated for 16 hrsat 37° C., 5% CO₂. Cell lysates were then prepared by removing the mediafrom the cells, washing them in 1×PBS, and adding 1 mL 1×PLB. Lysateswere then diluted 1:10 in 1×PBS with 0.1% gelatin. 40 μL of the dilutedlysate was then used in a TEV protease digestion as described above. 10μL of the digestion was mixed with 40 μL DMEM without phenol red, and 50μL assay reagent (previously described; 100 μM PBI-3939) added.Luminescence was measured as previously described (FIG. 73H).

3. For analysis in E. coli, E. coli cultures expressing the CP L27Vvariants were grown overnight at 30° C. These cultures were used (1:100diluted in LB+antibiotic) to make new starter cultures for eventualinduction. The starter cultures were incubated at 37° C. with shakingfor 2.5 hrs (OD₆₀₀ is approximately 0.5). Rhamnose (final concentrationof 0.2%) was added, the cultures moved to 25° C., and incubated withshaking for 18 hrs.

To create lysates, 50 μl 0.5× FASTBREAK™ Cell Lysis Reagent (PromegaCorp.) was added to 950 μL of induced cultures, and the mixturesincubated for 30 min at 22° C. 50 μL of the lysed culture was thendigested with TEV protease as described above and incubated at roomtemperature for 2 hrs.

For analysis, lysates were diluted 1:10,000 in HaloTag® MammalianPurification Buffer (Promega Corp.), and 50 μL assayed in 50 μL of assayreagent (as previously described; 100 μM PBI-3939). Basal and TEVinduced luminescence was measured at 5 min time points (FIG. 73F) andthe response (FIG. 73G) was determined as previously described.

FIGS. 73A-D show the basal luminescence of various CP-TEV protease L27Vconstructs expressed in wheat germ extract. FIG. 73E summarizes thederived CP variants that responded to TEV protease (response is folddecreased), indicating that the CP variants can be used as TEV sensors,i.e., they are indicative of “tether dependence.” Variants showing atleast a 1.2-fold change were further validated as signification usingStudent's Test (unpaired p values; confidence level of 0.03). Theseresults indicate that each CP variant is capable of generatingluminescence.

Various CP-TEV protease L27V constructs were expressed in HEK293 cells.The reverse transfection protocol, previously described was used totransfect 1 ng DNA/well with 1 μg of carrier DNA. Each cell sample wasgrown in 1 mL of media in a 12 well plate. Cell lysates were prepared byremoving media and adding 1 mL of 1×PLB. Samples were diluted 1:10 in1×PBS+0.1% gelatin. 40 μL of the dilution sample was set up for TEVdigestion. 10 μL of the digestion reaction was added to 40 μL of DMEMwithout phenol red and 50 μL of PBI-3939 as previously described. FIG.73H shows the luminescence of the various CP-TEV protease L27Vconstructs expressed in HEK293 cells.

The data in FIGS. 73A-H demonstrates that the L27V variant can becircularly permuted at various sites, and different sites have differentdependencies regarding tether length. The mammalian cell data and WheatGerm data show similar fold reduction with TEV cleavage. The CP L27Vvariants that are more dependent on the tether, i.e., are more sensitiveto TEV protease cleavage, are potential candidates for PCA. CP L27Vvariants that are less dependent on the tether may be potentialcandidates for self-complementation/dimerization assays.

Example 49 Protein Complementation Assays

Protein complementation assays (PCA) provide a means to detect theinteraction of two biomolecules, e.g., polypeptides. PCA utilizes twofragments of the same protein, e.g., enzyme, that when brought intoclose proximity with each other can reconstitute into a functional,active protein. An OgLuc variant of the present invention can beseparated into two fragments at a site(s) tolerant to separation. Then,each fragment of the separated OgLuc variant can be fused to one of apair of polypeptides of interest believed to interact, e.g., FKBP andFRB. If the two polypeptides of interest do in fact interact, the OgLucfragments then come into close proximity with each other to reconstitutethe functional, active OgLuc variant. The activity of the reconstitutedOgLuc variant can then be detected and measured using a native or knowncoelenterazine or a novel coelenterazine of the present invention. Inanother example, the split OgLuc variant can be used in a more generalcomplementation system similar to lac-Z (Langley et al., PNAS,72:1254-1257 (1975)) or ribonuclease S (Levit and Berger, J. Biol.Chem., 251:1333-1339 (1976)). Specifically, an OgLuc variant fragment(designated “A”) known to complement with another OgLuc variant fragment(“B”) can be fused to a target protein, and the resulting fusion can bemonitored via luminescence in a cell or a cell lysate containingfragment B. The source of fragment B could be the same cell (either inthe chromosome or on another plasmid), or it could be a lysate orpurified protein derived from another cell. This same fusion protein(fragment A) could be captured or immobilized using a fusion betweenfragment B and a polypeptide such as HALOTAG® capable of attachment to asolid support. Luminescence can then be used to demonstrate successfulcapture or to quantitate the amount of material captured. Methods forprotein complementation can be carried out according to U.S. PublishedApplication No. 2005/0153310, incorporated herein by reference.

1. 9B8 opt PCA constructs were made as follows:

(SEQ ID NOs: 331 and 332) -p9B8PCA 1/4 =pF5A/Met-[9B8 opt (51-169)]-GGGGSGGGSS-FRB & (SEQ ID NOs: 337 and 338)pF5A/FKBP-GGGSSGGGSGA-[9B8 opt (1-50)] (SEQ ID NOs: 331 and 332)-p9B8PCA 1/2 = pF5A/Met-[9B8 opt (51-169)]-GGGGSGGGSS-FRB &(SEQ ID NOs: 333 and 334) pF5A/[9B8 opt (1-50)]-GGGGSGGGSS-FRB(SEQ ID NOs: 333 and 334) -p9B8PCA 2/3 =pF5A/[9B8 opt (1-50)]-GGGGSGGGSS-FRB & (SEQ ID NOs: 335 and 336)pF5A/FKBP-GGGSSGGGSG-[9B8 opt (51-169)] (SEQ ID NOs: 335 and 336)-p9B8PCA 3/4 = pF5A/FKBP-GGGSSGGGSG-[9B8 opt (51-169)] &(SEQ ID NOs: 337 and 338) pF5A/FKBP-GGGSSGGGSG-[9B8 opt (1-50)]

The PCA constructs were transfected into HEK293 cells (15,000cells/well) into a 96-well plate using FUGENE® HD according to themanufacturer's instructions. The cells were then incubated overnight at37° C., 5% CO₂. After transfection, the media on the cells was replacedwith CO₂-independent media with 10% FBS. Assay reagent with 20 μMPBI-3939 was then added, and luminescence measured on a Varioskan Flashat 28° C. 100 μM rapamycin was then added to the wells, and luminescencecontinuously measured for 1 hr. Fold response was calculated by dividingall luminescence of a given well by the pre-rapamycin treatmentluminescence for the same well (FIG. 74).

2. To demonstrate the use of the OgLuc variants in PCA, the L27V02Avariant fragments were complementated with FKBP or FRB, and theinteraction between FKBP and FRB measured.

Table 42 lists the various protein complementation (PCA) constructs madeand tested. “2/3” designates the variant complementation pairs where 1)the “old” C-terminus of L27V02A (“old”=original C-terminus of L27V02A)is the C-terminal partner of FKBP; and 2) the “old” N-terminus ofL27V02A is the N-terminal partner of FRB. “1/4” designates the variantpairs where 1) the “old” N-terminus of L27V02A is the C-terminal partnerof FKBP; and 2) the “old” C-terminus of L27V02A is the N-terminalpartner of FRB. For all constructs, FKBP was located at the N-terminusof the L27V02A fragment, and FRB was located at the C-terminus of theL27V02A fragment. For example, PCA constructs were made with split sitesat position 157 (see Table 42, “2/3” and “1/4” #s 11 and 12 (SEQ ID NOs:288-295)), 103 (see Table 42, “2/3” and “1/4” #s 9 and 10 (SEQ ID NOs:296-303)), and 84 (see Table 42, “2/3” and “1/4” #s 7 and 8 (SEQ ID NOs:304-315)). Other PCA constructs were made (SEQ ID NOs: 343-426 and428-440) (See Table 21)

TABLE 42 “⅔” “¼” 1. FKBP-L27V 6-169 1. FKBP-L27V 1-5 2. L27V 1-5-FRB 2.L27V 6-169-FRB 3. FKDP-L27V 12-169 3. FKBP-L27V 1-11 4. L27V 1-11-FRB 4.L27V 12-169-FRB 5. FKBP-L27V 55-169 5. FKBP-L27V 1-54 6. L27V 1-54-FRB6. L27V 55-169-FRB 7. FKBP-L27V 84-169 7. FKBP-L27V 1-83 8. L27V1-83-FRB 8. L27V 84-169-FRB 9. FKBP-L27V 103-169 9. FKBP-L27V 1-102 10.L27V 1-102-FRB 10. L27V 103-169-FRB 11. FKBP-L27V 157-169 11. FKBP-L27V1-156 12. L27V 1-156-FRB 12. L27V 157-169-FRB 1. FKBP-L27V 6-169 1.FKBP-L27V 1-5 2. L27V 1-5-FRB 2. L27V 6-169-FRB 3. FKBP-L27V 12-169 3.FKBP-L27V 1-11 4. L27V 1-11-FRB 4. L27V 12-169-FRB 5. FKBP-L27V 55-1695. FKBP-L27V 1-54 6. L27V 1-54-FRB 6. L27V 55-169-FRB 7. FKBP-L27V84-169 7. FKBP-L27V 1-83 8. L27V 1-83-FRB 8. L27V 84-169-FRB 9.FKBP-L27V 103-169 9. FKBP-L27V 1-102 10. L27V 1-102-FRD 10. L27V103-169-FRB 11. FKBP-L27V 157-169 11. FKBP-L27V 1-156 12. L27V 1-156-FRB12. L27V 157-169-FRB

pCA constructs   SEQ ID NO: 343 (pCA 31 pCA L27V02A 45-169 FRB)   SEQ IDNO: 344 (pCA 31 pCA L27V02A 45-169 FRB)   SEQ ID NO: 345 (pCA 32 FKBPL27V02A 1-44)   SEQ ID NO: 346 (pCA 32 FKBP L27V02A 1-44)   SEQ ID NO:347 (pCA 33 pCA L27V02A 45-169 FRB)   SEQ ID NO: 348 (pCA 33 pCA L27V02A45-169 FRB)   SEQ ID NO: 349 (pCA 34 FKBP 1-45 L27V02A)   SEQ ID NO: 350(pCA 34 FKBP 1-45 L27V02A)   SEQ ID NO: 351 (pCA 35 pCA L27V02A 100-169FRB)   SEQ ID NO: 352 (pCA 35 pCA L27V02A 100-169 FRB)   SEQ ID NO: 353(pCA 36 FKBP L27V02A 1-99)   SEQ ID NO: 354 (pCA 36 FKBP L27V02A 1-99)  SEQ ID NO: 355 (pCA 37 L27V02A 101-169 FRB)   SEQ ID NO: 356 (pCA 37L27V02A 101-169 FRB)   SEQ ID NO: 357 (pCA 38 FKBP 1-100 L27V02A)   SEQID NO: 358 (pCA 38 FKBP 1-100 L27V02A)   SEQ ID NO: 359 (pCA 39 L27V02A102-169 FRB)   SEQ ID NO: 360 (pCA 39 L27V02A 102-169 FRB)   SEQ ID NO:361 (pCA 40 FKBP L27V02A 1-101)   SEQ ID NO: 362 (pCA 40 FKBP L27V02A1-101)   SEQ ID NO: 363 (pCA 41 L27V02A 143-169 FRB)   SEQ ID NO: 364(pCA 41 L27V02A 143-169 FRB)   SEQ ID NO: 365 (pCA 42 FKBP 1-142L27V02A)   SEQ ID NO: 366 (pCA 42 FKBP 1-142 L27V02A)   SEQ ID NO: 367(pCA 43 L27V02A 145-169 FRB)   SEQ ID NO: 368 (pCA 43 L27V02A 145-169FRB)   SEQ ID NO: 369 (pCA 44 FKBP 1-144 L27V02A)   SEQ ID NO: 370 (pCA44 FKBP 1-144 L27V02A)   SEQ ID NO: 371 (pCA 45 L27V02A 147-169 FRB)  SEQ ID NO: 372 (pCA 45 L27V02A 147-169 FRB)   SEQ ID NO: 373 (pCA 46FKBP-L27V02A 1-146)   SEQ ID NO: 374 (pCA 46 L27V02A-FKBP 1-143)   SEQID NO: 375 (pCA 47 L27V02A 148-169 FRB)   SEQ ID NO: 376 (pCA 47 L27V02A148-169 FRB)   SEQ ID NO: 377 (pCA 48 FKBP-L27V02A 1-147)   SEQ ID NO:378 (pCA 48 FKBP-L27V02A 1-147)   SEQ ID NO: 379 (pCA 49 L27V02A 156-169FRB)   SEQ ID NO: 380 (pCA 49 L27V02A 156-169 FRB)   SEQ ID NO: 381 (pCA50 FKBP-L27V02A 1-155)   SEQ ID NO: 382 (pCA 50 FKBP-L27V02A 1-155)  SEQ ID NO: 383 (pCA 51 L27V02A 158-169 FRB)   SEQ ID NO: 384 (pCA 51L27V02A 158-169 FRB)   SEQ ID NO: 385 (pCA 52 FKBP 1-157 L27V02A)   SEQID NO: 386 (pCA 52 FKBP 1-157 L27V02A)   SEQ ID NO: 387 (pCA 53 L27V02A166-169 FRB)   SEQ ID NO: 388 (pCA 53 L27V02A 166-169 FRB)   SEQ ID NO:389 (pCA 54 FKBP L27V02A 1-165)   SEQ ID NO: 390 (pCA 54 FKBP L27V02A1-165)   SEQ ID NO: 391 (pCA 55 FKBP L27V02A 1-47)   SEQ ID NO: 392 (pCA55 FKBP L27V02A 1-47)   SEQ ID NO: 393 (pCA 56 L27V02A 48-169-FRB)   SEQID NO: 394 (pCA 56 L27V02A 48-169-FRB)   SEQ ID NO: 395 (pCA 57 FKBPL27V02A 1-49)   SEQ ID NO: 396 (pCA 57 FKBP L27V02A 1-49)   SEQ ID NO:397 (pCA 58 pCA L27V02A 50-169 FRB)   SEQ ID NO: 398 (pCA 58 pCA L27V02A50-169 FRB)   SEQ ID NO: 399 (pCA 59 FKBP L27V02A 1-82)   SEQ ID NO: 400(pCA 59 FKBP L27V02A 1-82)   SEQ ID NO: 401 (pCA 60 L27V02A 83-169-FRB)  SEQ ID NO: 402 (pCA 60 L27V02A 83-16-FRB)   SEQ ID NO: 403 (pCA 61FKBP L27V02A 1-84)   SEQ ID NO: 404 (pCA 61 FKBP L27V02A 1-84)   SEQ IDNO: 405 (pCA 62 L27V02A 85-169-FRB)   SEQ ID NO: 406 (pCA 62 L27V02A85-169-FRB)   SEQ ID NO: 407 (pCA 63 FKBP L27V02A 1-122)   SEQ ID NO:408 (pCA 63 FKBP L27V02A 1-122)   SEQ ID NO: 409 (pCA 64 L27V02A123-169-FRB)   SEQ ID NO: 410 (pCA 64 L27V02A 123-169-FRB)   SEQ ID NO:411 (pCA 65 FKBP L27V02A 1-123)   SEQ ID NO: 412 (pCA 65 FKBP L27V02A1-123)   SEQ ID NO: 413 (pCA 66 L27V02A 124-169 FRB)   SEQ ID NO: 414(pCA 66 L27V02A 124-169 FRB)   SEQ ID NO: 415 (pCA 67 L27V02A 1-168)  SEQ ID NO: 416 (pCA 67 L27V02A 1-168)   SEQ ID NO: 417 (pCA 67 L27V02A1-168)   SEQ ID NO: 418 (*pCA 68 L27V02A 169 FRB)   SEQ ID NO: 419 (*pCA68 L27V02A 169 FRB)   SEQ ID NO: 420 (pCA 69 FKBP L27V02A 1-166)   SEQID NO: 421 (pCA 69 FKBP L27V02A 1-166)   SEQ ID NO: 422 (*pCA 70 L27V02A167-169 FRB)   SEQ ID NO: 423 (*pCA 70 L27V02A 167-169 FRB)   SEQ ID NO:424 (pCA 71 FKBP L27V02A 1-164)   SEQ ID NO: 425 (pCA 71 FKBP L27V02A1-164)   SEQ ID NO: 426 (pCA 72 L27V02A 165-169 FRB)   SEQ ID NO: 428(pCA 72 L27V02A 165-169 FRB)   SEQ ID NO: 429 (pCA 73 FKBP L27V02A1-162)   SEQ ID NO: 430 (pCA 73 FKBP L27V02A 1-162)   SEQ ID NO: 431(pCA 74 L27V02A 163-169 FRB)   SEQ ID NO: 432 (pCA 74 L27V02A 163-169FRB)   SEQ ID NO: 433 (pCA 75 FKBP L27V02A 1-160)   SEQ ID NO: 434 (pCA75 FKBP L27V02A 1-160)   SEQ ID NO: 435 (pCA 76 L27V02A 161-169 FRB)  SEQ ID NO: 436 (pCA 76 L27V02A 161-169 FRB)   SEQ ID NO: 437 (pCA 77FKBP L27V02A 1-158)   SEQ ID NO: 438 (pCA 77 FKBP L27V02A 1-158)   SEQID NO: 439 (pCA 78 L27V02A 159-169 FRB)   SEQ ID NO: 440 (pCA 78 L27V02A159-169 FRB)

The complementation pairs described in Table 42 were cloned into thepF4Ag vector as previously described. The PCA constructs (900 μL) werethen expressed in rabbit reticulocyte lysate (RRL; Promega Corp.) orwheat germ extract (Promega Corp.) following the manufacture'sinstructions. 1.25 μL of the expression reactions for each PCA pair weremixed with 10 μL of 2× Binding Buffer (100 mM HEPES, 200 mM NaCl, 0.2%CHAPS, 2 mM EDTA, 20% glycerol, 20 mM DTT, pH 7.5) and 7.5 μL water, and18 μL transferred to wells of a 96-well plate. 2 μL of 5 μM Rapamycin(final concentration 0.5 μM) was added and incubated for 10 min at roomtemperature.

Following incubation, 100 μL of PBI-3939 (50× stock diluted to 1× inassay buffer) and incubate for 3 min at room temperature. Luminescencewas measured as previously described (FIG. 76A-B: wheat germ; FIG.76C-D: rabbit reticulocyte; FIG. 76E-F: cell free system [which system?WG or RRL?]; FIG. 76G: HEK293 cells).

FIG. 76A-G show the luminescence of various protein complementation(PCA) L27V pairs: one L27V fragment of each pair was fused to eitherFKBP or FRB using a 2/3 configuration (FIGS. 76A and 76C) or a 1/4configuration (FIGS. 76B and 76D) as described, and the interaction ofFKBP and FRB monitored in wheat germ extract (FIGS. 76A and 76B) andrabbit reticulocyte lysate (RRL) (FIGS. 76C and 76D); and theluminescence of various protein complementation (PCA) negative controls(FIG. 76E). The luminescence of various protein complementation L27Vusing a 1/4 configuration in a cell free system (RRL) (FIG. 76F) andHEK293 cells (FIG. 76G) was measured. The data in FIGS. 76A-Gdemonstrates that a variety of different deletions, i.e., smallfragments of the L27V variant, are functional.

3. To demonstrate the use of the PCA constructs for cell-based PCA, theconstructs were transfected into HEK293 cells and assayed with PBI-4377.Plasmid DNA (5 ng) from each PCA pair (6, 12, 55, 84, and 103) weremixed with 40 ng carrier DNA (pGEM-3fz) and 5 OPTI-MEM® and incubated atroom temperature for 5 min. FUGENE® HD (0.15 μL) was then added andagain incubated at room temperature for 15 min. The DNA transfectionmixtures were added to 100 μL HEK293 cells (1.5×10⁵ cells/mL) in DMEMwith 10% FBS (no antibiotics), transferred to wells of a 96-well plate,and incubated overnight at 37° C., 5% CO₂.

After transfection, the media was removed and replaced withCO₂-independent media with 20 μM or 50×PBI-4377 and incubated at 37° C.without CO₂ regulation for 2 hrs. Luminescence was measured, 10 μLrapamycin added, and luminescence measured again every 2 min for 2 hrs(FIGS. 76A-C).

4. To demonstrate the use of the PCA constructs to identify inhibitorsof protein-protein interactions, the constructs described in #2 of thisexample were used.

The complementation pairs, 103 “2/3”, 157 “2/3”, 103 “1/4” and 157 “1/4”described in Table 42 were cloned into the pF4Ag vector as previouslydescribed. The PCA constructs (25 μL) were then expressed in rabbitreticulocyte lysate (RRL; Promega Corp.) following the manufacture'sinstructions. 1.25 μL of the expression reactions for each PCA pair weremixed with 10 μL of 2× Binding Buffer (100 mM HEPES, 200 mM NaCl, 0.2%CHAPS, 2 mM EDTA, 20% glycerol, 20 mM DTT, pH 7.5) and 7.5 μL water, and16.2 μL transferred to wells of a 96-well plate. Rapamycin was examinedwith various amounts of FK506. To the reactions, the FRB-FKBP bindinginhibitor, FK506 (10×) was added, and the reactions incubated at roomtemperature for 10 min. 15 nM rapamycin (10× stock solution) was addedto get a final concentration of 1.5 nM rapamycin and incubated for 2 hrsat room temperature. Following incubation, 100 μL of PBI-3939 (50× stockdiluted to 1× in assay buffer) and incubated for 3 min at roomtemperature. Luminescence was measured on a GLOMAX® luminometer. FIG. 77demonstrates that the PCA constructs disclosed herein can be used toidentify inhibitors of protein-protein interactions.

5. To demonstrate the use of the PCA constructs in a lytic format, thecomplementation pairs, 103 “2/3”, 157 “2/3”, and 103 “1/4” weretransfected into HEK293 cells and assayed with PBI-3939. 0.5 ng plasmidfrom each PCA pair was mixed with 5 μL OPTI-MEM® and 49 ng pGEM-3zf(Promega Corp.). The sample mixture was incubated at room temperaturefor 5 min. 0.15 μL FUGENE® HD was then added to the sample mixture andincubated at room temperature for 15 min. 100 μL of HEK293 cells in DMEMwith 10% FBS (no antibiotics) at a concentration of 1.5×10⁵ cells/mL wasadded to each sample mixture. The cell sample was then transferred to awell of a 96-well plate and incubated at 37° C., 5% CO₂ overnight.

The next day, 11.1 μL of 10 μM Rapamycin (Final concentration 1 μM) wasadded to half of the wells and 11.1 μL water was added to other half ofthe wells. The 96-well plates were incubated at 37° C. for 1 hr. 100 μLof assay reagent+PBI-3939 (2 μL 50×PBI-3939 mixed with 98 μL assayreagent, previously described) was added to each well and the plateswere incubated at 37° C. for 4 min. Luminescence was measured on aGLOMAX® luminometer at 37° C. with 0.5 s integration time and 1 read.(FIG. 76H).

Example 50 OgLuc cAMP Biosensor

The OgLuc variants of the present invention can be linked to lightoutput not only through concentration, but also through modulation ofenzymatic activity. For example, a cAMP biosensor can be developed byincorporating a cAMP-binding domain from Protein Kinase A into acircularly permuted OgLuc variant. An OgLuc variant of the presentinvention can be circularly permuted at a site(s) tolerable to suchpermutation by methods known in the art (e.g., U.S. PublishedApplication No. 2005/0153310). The resulting circularly permuted OgLucvariant chimeric protein can function as an intracellular biosensor forcAMP when expressed in mammalian cells. Upon binding of cAMP to thebiosensor, the biosensor undergoes a conformational change that createsan active luciferase enzyme. Treating the cells with forskolin, anactivator for adenylate cyclase, should result in an increase inluminescence with increasing concentrations of forskolin. Similarbiosensors for targets including but not limited to calcium (Ca+2),cGMP, and proteases such as caspases and tobacco etch virus (TEV) can bedeveloped by incorporating the appropriate binding domain or cleavagesite for each into a circularly permuted OgLuc variant.

The utility of OgLuc as a biosensor was demonstrated by analysis ofvariant 9B8 opt in the context of a cAMP sensor. Circularly permutedconstructs containing the RIIβB subunit of Protein Kinase-A flanked byOgLuc variant sequences were made and expressed in a cell free system asdescribed in described in PCT application PCT/US2007/008176, except thesites for circular permutation were chosen as described below. Thenascent protein was assayed in the presence and absence of cAMP.Response to cAMP is defined as the ratio of activity (+) cAMP/(−) cAMP.

A structural model for OgLuc has been created, based on similarities tocertain fatty acid binding proteins of known structure, previouslydescribed in PCT/US2010/33449. The model predicts an ordered sequence ofthe standard protein structural motifs; α-helix and β-sheet. The regionsthat transition between these structural elements as circularpermutation sites were chosen (see Table 43).

1. The template for expression of the biosensor constructs consisted of:C-terminal OgLuc sequence-RIIβB sequence-N-terminal OgLuc sequence inplasmid pF5 (Promega Corp.). The TNT® T7 Coupled Wheat Germ ExtractSystem (Promega Part #L4140) was used to translate the construct. TheTNT® Wheat Germ Extract Reaction included 25 μl, TNT® Wheat Germ Extract(L411A), 2 μL TNT® Reaction Buffer (L462A), 1 μL Amino Acid Mixture,Complete (L446A), 1 μL RNasin® (40 U/μL) (N2615), 1 μL TNT® T7 RNAPolymerase (L516A), 1.0 μg DNA template and Nuclease-Free Water to bringthe total volume to 50 μL. The reaction mixture was incubated at 30° C.for 120 min.

An OgLuc activity assay was performed by adding to the 50 μL OgLuctranslation mixture, 50 μL OgLuc Glo Reagent (100 mM MES (pH 6.0), 1 mMCDTA, 150 mM KCl, 35 mM thiourea, 2 mM DTT, 0.25% TERGITOL® NP-9 (v/v),0.025% MAZU® DF 204, and 20 μM PBI-3939) with or without 100 μM cAMP,and performing a kinetic read for 30 min (TECAN® INFINITE® F500 PlateReader). Response is determined by dividing the luminescence generatedby the biosensor with cAMP by the luminescence generated by thebiosensor without cAMP (Table 43).

TABLE 43 Response of Circularly-Permuted OgLuc Biosensors to cAMPCP-SITE RESPONSE 27 2.6X 51 2.2X 84 1.5X 122 4.3X 147 1.9X 157 5.6X

2. A cAMP biosensor of 9B8opt circularly permuted at the CP-site 51 wascreated as described in 1. The biosensor was then transfected intoHEK293 cells (15,000 cells/well) using FUGENE® HD according to themanufacturer's instructions into a 96-well plate, and incubatedovernight at 37° C., 5% CO₂. After transfection, the media was removedand replaced with CO₂-independent media with 10% FBS. The cells werethen incubated for 2 hrs at 37° C., 5% CO₂ after which varying amountsof FSK were added. The cells were then again incubated for 3 hrs at 37°C., 5% CO₂. 6 μM PBI-3939 was then added, and luminescence measuredafter 13 min (FIG. 78).

3. Circularly permuted (“CP”; e.g., CP6 refers to the old residue 6being new residue 1 after Met) and Straight Split (“SS”; e.g., SS6refers to a sensor orientated as follows: OgLuc (1-6)-RIIβb binding site(SEQ ID NOs: 441 and 442)-OgLuc (7-169)) versions of L27V were used ascAMP biosensors (SEQ ID NOs: 467-574). CP (SEQ ID NOs: 467-498 and555-574) and SS (SEQ ID NOs: 499-554) versions of the L27V variant werederived as previously described and expressed in expressed in rabbitreticulocyte lysate (RRL; Promega Corp.) following the manufacture'sinstructions. The linker sequence between the C-terminus of the RIIβbbinding site and OgLuc luciferase sequence wasGGGTCAGGTGGATCTGGAGGTAGCTCTTCT (SEQ ID NO: 575). The linker sequencebetween the N-terminus of the RIIβb binding site and OgLuc luciferasesequence was AGCTCAAGCGGAGGTTCAGGCGGTTCCGGA (SEQ ID NO: 576) 3.75 μL ofthe expression reactions were mixed with 1.25 μL 4×cAMP (finalconcentration 1 nM-0.1 mM), and incubated at room temperature for 15min. Following incubation, 100 μL of PBI-3939 (50× stock diluted to 1×in assay buffer) and incubated for 3 min at room temperature.Luminescence was measured on a GLOMAX® luminometer (FIGS. 79A-B).Luminescence was also measured for CP and SS versions of the L27Vvariant expressed in HEK293 cells and treated with forskolin aspreviously described (FIGS. 79C-D). FIGS. 79A-D demonstrates thatcircularly permuted and straight split versions of the OgLuc variantsdisclosed herein can be used as biosensors.

Example 51 Subcellular Distribution and Localization

To analysis subcellular distribution, U20S cells were plated at 2×10⁴cells/cm² into glass-bottom culture dishes in McCoy's 5A media(Invitrogen) containing 10% FBS. The cells were then incubated for 24hrs at 37° C., 5% CO₂. Cells were then transfected with 1/20 volumetransfection mixture (FUGENE® HD and pF5A-CMV-L27V (the L27V variant(SEQ ID NO: 88) cloned into the pF5A vector with CMV promoter (PromegaCorp.)) or pGEM3ZF (Promega Corp.; negative control)) and incubated for24 hrs at 37° C., 5% CO₂. Following incubation, the cell media wasreplaced with CO₂-independent media with 0.5% FBS and 100 μM PBI-4378.After a 30 min incubation at 37° C., unfiltered images were captured onan Olympus LV200 bioluminescence microscope using a 60× objective (FIGS.80A-B) for 25, 100, 1000, and 5000 ms.

To analyze subcellular localization, N-terminal L27V fusions with theGPCR AT1R (Angiotensin type 1 receptor (SEQ ID NOs: 459 and 460)) withIL-6 secretion sequence (SEQ ID NOs: 461 and 462) or the transcriptionfactor, Nrf2 (SEQ ID NO: 317), were made using a GSSG linker (SEQ IDNOs: 457 and 458) and transfected into U20S cells as described above(FIGS. 81A-C). FIG. 81C (“GPRC”) shows expression of a construct wherethe IL6 signal sequence is upstream of the L27V variant sequence and theAT1R is downstream of the L27V variant sequence. The L27V variant alonewas also transfected (“Unfused”). After a 24 hr incubation at 37° C., 5%CO₂, cell media was replaced with CO₂-independent media with 0.5% FBSand equilibrated for 1 hr at 37° C. in a non-CO₂-regulated atmosphere.An equal volume of media+200 μM PBI-3939 was then added, and unfilteredimages were captured immediately on an Olympus LV200 bioluminescencemicroscope using a 60× or 150× objective (FIGS. 81A-C). For cellsexpressing L27V alone, PBI-3939 was washed off the cells immediatelybefore image capture.

Example 52 Monitoring Intracellular Signal Pathways

This example provides two examples of the novel luciferase being used tomonitor intracellular signal pathways at the protein level (as opposedto the response element examples which represent transcriptionalactivation). The variant 9B8opt (SEQ ID NO: 24) was fused to either 1 kB(Gross et al., Nature Methods 2(8):607-614 (2005)) (at the C-terminus,i.e., N-IkB-(9B8opt)-C)) or ODD(oxygen-dependent degradation domain ofHif-1-α (Moroz et al., PLoS One 4(4):e5077 (2009)) (at the N-terminus,i.e., N-(9B8opt)-ODD-C)). IKB is known to be degraded in cells uponstimulation with TNFα; therefore, the IKB-(9B8opt) construct could beused as a live cell TNFα sensor. ODD (Hif-1-α) is known to accumulateinside cells upon stimulation with compounds that induce hypoxia;therefore, the ODD-(9B8opt) construct could be used as a live cellhypoxia sensor.

Constructs containing fusions with 1 kB or ODD with 9B8opt (pF5A) wereexpressed in HEK293 cells via reverse transfection (5 ng (IkB) or 0.05ng (ODD) DNA (mixed with carrier DNA to give a total of 50 ng)) aspreviously described and incubated for 24 hrs at 37° C., 5% CO₂. Aftertransfection, the media was replaced with fresh CO₂-independent mediacontaining 0.5% FBS and 20 μM PBI-4377 and allowed to equilibrate for 4hrs at 37° C., atmospheric CO₂. Cells were then exposed to a stimulus:TNFα for IkB fusion expressing cells and phenanthroline for ODD fusionexpressing cells. DMSO (vehicle) was added to control cells. For theTNFα/IKB samples, 100 μg/mL cycloheximide was added approximately 15 minprior to adding the stimulus to prevent synthesis of new protein. At theindicated time points following treatment, cells were assayed forluminescence. For data normalization, the RLU of each sample at a giventime point were divided by the RLU from the same sample immediatelyafter stimulation. Fold response for each sensor was then determined(FIGS. 82A-C).

B. L27V was used to monitor the oxidative stress signal pathways at theprotein level. L27V or Renilla luciferase (Rluc) was fused toNrf2/NFE2L2 in a pF5K expression vector (at the C-terminus; i.e.,N-Nrf2-(L27V)-C or N-Nrf2-(Rluc)-C). Keap1 is a negative regulator ofNrf2 (SEQ ID NO: 217). In order to faithfully represent regulation ofNrf2-L27V02 protein levels, Keap1, was co-expressed to keep Nrf2 levelslow (via ubiquitination).

Nrf2-L27V or Nrf2-Rluc (5 ng, pF5K) and a HALOTAG®-Keap 1 fusion protein(pFN21-HT7-Keapl (SEQ ID NO: 316); 50 ng) were expressed in HEK293 cellsby transfection of the cells at the time of seeding into the 96-wellplates as previously described and incubated for 24 hrs at 37° C., 5%CO₂. After transfection, the media was replaced with freshCO₂-independent media with 0.5% FBS and 20 μM PBI-4377 for L27V or 20 μMENDUREN™ (Promega Corp.) for Renilla luciferase, and the cellsequilibrated for 4 hrs at 37° C. under atmospheric CO₂. For kineticanalysis, 20 μM D,L sulforaphane or vehicle (DMSO) were used. In FIG.83A, luminescence was measured as previously described at the indicatedtime points following treatment. For data normalization, theluminescence of each sample at a given time point was divided by theluminescence from the same sample immediately after stimulation (FIGS.83B-C).

C. A comparison of the response of the Nrf2 sensor described in B andNrf2(ARE)-Luc2P reporter (Promega Corp.) was performed. Both the Nrf2sensor and reporter were screened as described in section B above. Forthe firefly (Luc2P) reporter gene assay, the ONE-GLO™ assay reagent wasused. FIGS. 84A-B provides the normalized response of Nrf2-L27V at 2 hrsand Nrf2(ARE)-Luc2P at 16 hrs

Example 53 Evaluation of OgLuc Variant as Bioluminescent Reporter withBRET

Bioluminescence resonance energy transfer (BRET) allows monitoring ofprotein-protein interactions. The intramolecular energy transfer wasexamined between IV and a HT7 fusion partner where HT7 was previouslylabeled with a fluorophore, i.e., TMR (excitation/emission (ex/em)wavelength=555/585 nm) or Rhodamine 110 (excitation/emissionwavelength=502/527 nm). 50 μL of a bacterial cell lysate containing theIV-HT7 fusion protein of Example 34 was incubated with or without0.001-10 μM fluorophore ligand for 1 hr at room temperature. After theincubation, 50 μL of RENILLA-GLO™, which contains 22 μMcoelenterazine-h, was added to 50 μL of the enzyme-ligand mixture, andthe emission spectrum was recorded at 5 min. Example spectra of IV-HT7with TMR (FIG. 83A) or Rhodamine 110 (“Rhod110”) (FIG. 85B) are shownindicating BRET was greater when the ex/em of the ligand was closer tothe 460 nm luminescent peak of OgLuc, i.e., greater with Rhodamine 110.This data shows that intramolecular energy transfer can occur betweenOgLuc variants and a fluorophore on a fusion protein. Three differentcontrols were used for comparison (data not shown): 1) a non-HT fusion,2) a HT-fusion that was not labeled with a HT ligand, and 3) a labeledHT-fusion that was proteolytically cleaved at a TEV site between OgLucand FIT (which indicated the involvement of proximity/distance). BRETwas not observed in the three different controls suggesting that HT wasinvolved to achieve BRET. BRET was greater for C1+A4E and IV with aC-terminal HT7 compared to N-terminal HT7.

Example 54 Protein Proximity Assays for Live Cells or Lytic Formats

In one example, circularly permuted (CP) or straight split (SS) OgLucfusion proteins is applied to measurements of protein proximity. OgLucis permuted or split via insertion of a protease substrate amino acidsequence (e.g., TEV) to generate low bioluminescence. The inactiveluciferase is tethered (e.g., via genetic fusion) to a monitor protein.A potential interacting protein is tethered (e.g., via genetic fusion)to a protease (e.g., TEV). When the two monitor proteins interact or arein sufficient proximity (e.g., via a constitutive interaction, a drugstimulus or a pathway response), the luciferase is cleaved to generateincreased bioluminescence activity. The example may be applied tomeasurements of protein proximity in cells or in biochemical assays.Furthermore, the high thermal stability of an OgLuc variant luciferasecould enable measurements of antibody-antigen interactions in lysedcells or biochemical assays.

Example 55 Bioluminescent Assays

1. To demonstrate the use of an OgLuc variant in a bioluminescent assayto detect caspase-3 enzyme, the 9B8 opt variant was used in abioluminescent assay using a pro-coelenterazine substrate comprising theDEVD caspase-3 cleavage sequence. Purified caspase-3 enzyme was mixedwith an E. coli lysate sample expressing the variant 9B8 opt, which wasprepared as described in Example 27, and diluted 10-fold in a buffercontaining 100 mM MES pH 6.0, 1 mM CDTA, 150 mM KCl, 35 mM thiourea, 2mM DTT, 0.25% TERGITOL® NP-9 (v/v), 0.025% MAZU® DF 204, with or without23.5 μM z-DEVD-coelenterazine-h in 100 mM HEPES pH 7.5. The caspase-3enzyme was incubated with the lysate sample for 3 hrs at roomtemperature, and luminescence detected on a Turner MODULUS™ luminometerat various time points. A sample containing only bacterial lysate and asample containing only caspase-3 were used as controls. Three replicateswere used. FIG. 86 and Table 44 demonstrate that 9B8 opt, and thus otherOgLuc variants of the present invention, can be used in a bioluminescentassay with a pro-coelenterazine substrate to detect an enzyme ofinterest.

TABLE 44 Average luminescence in RLU generated from bacterial lysatesexpressing the 9B8 opt variant incubated with or without purifiedcaspase-3 using z-DEVD-coelenterazine-h as a substrate. time (min) nocaspase (RLU) + caspase (RLU) 5 26,023 25,411 15.3 7,707 36,906 29.94,013 41,854 60.9 2,305 43,370 190.3 1,155 42,448

2. The L27V variant was used in a bioluminescent assay using apro-coelenterazine substrate comprising the DEVD caspase-3 cleavagesequence. Purified caspase-3 enzyme (1 mg/mL) in 100 mM MES pH 6 (50 μL)was mixed with 227 nM L27V02 variant and 47 μM PBI-3741(z-DEVD-coelenterazine-h) in assay buffer (50 μL). Reactions wereincubated for 3 hrs at room temperature, and luminescence detected aspreviously described. The assay with the L27V variant was compared to afirefly luciferase version of the assay, CASPASE-GLO® 3/7-Assay system(Caspase-Glo; Promega Corp.). Table 45 demonstrate that L27V variant,and thus other OgLuc variants of the present invention, can be used in abioluminescent assay with a pro-coelenterazine substrate to detect anenzyme of interest.

TABLE 45 (+) caspase +/− (−) caspase +/− L27V 11,532 93 803 25Caspase-Glo 15,156,567 793,981 302 5

Example 56 Immunoassays

The OgLuc variants of the present invention are integrated into avariety of different immunoassay concepts. For example, an OgLuc variantis genetically-fused or chemically conjugated to a primary or secondaryantibody to provide a method of detection for a particular analyte. Asanother example, an OgLuc variant is genetically-fused or chemicallyconjugated to protein A, protein G, protein L, or any other peptide orprotein known to bind to Ig fragments, and this could then be used todetect a specific antibody bound to a particular analyte. As anotherexample, an OgLuc variant is genetically-fused or chemically conjugatedto streptavidin and used to detect a specific biotinylated antibodybound to a particular analyte. As another example, complementaryfragments of an OgLuc variant are genetically-fused or chemicallyconjugated to primary and secondary antibodies, where the primaryantibody recognizes a particular immobilized analyte, and the secondaryantibody recognizes the primary antibody, all in an ELISA-like format.The OgLuc variant activity, i.e., luminescence, is reconstituted in thepresence of immobilized analyte and used as a means to quantify theanalyte.

As another example, complementary fragment's of an OgLuc variant can befused to two antibodies, where one antibody recognizes a particularanalyte at one epitope, and the second antibody recognizes the analyteat a separate epitope. The OgLuc variant activity would be reconstitutedin the presence of analyte. The method would be amenable to measurementsof analyte quantification in a complex milieu such as a cell lysate orcell medium. As another example, complementary fragments of an OgLucvariant can be fused to two antibodies, where one antibody recognizes aparticular analyte regardless of modification, and the second antibodyrecognizes only the modified analyte (for example, followingpost-translational modification). The OgLuc variant activity would bereconstituted in the presence of analyte only when it is modified. Themethod would be amenable to measurements of modified analyte in acomplex milieu such as a cell lysate. As another example, an OgLucvariant can be conjugated to an analyte (e.g., prostaglandins) and usedin a competitive sandwich ELISA format.

Example 57 Dimerization Assay

This example demonstrates that full-length circularly permuted OgLucvariants can be fused to respective binding partners, e.g., FRB andFKBP, and used in a protein complementation-type assay. The keydifference between the method disclosed herein and traditional proteincomplementation is that there was no complementation, but rather therewas dimerization of two full length enzymes, e.g., circularly permutedOgLuc variants.

Briefly, the circularly permuted reporter proteins similarly configuredfor low activity were fused to both of the fusion protein partners (SeeFIG. 87A). For example, each fusion partner may be linked to identicallystructured, permuted reporters. Interaction of the fusion partnersbrought the permuted reporters into close proximity, thereby allowingreconstitution of a hybrid reporter having higher activity. The newhybrid reporter included portions of each of the circularly permutedreporters in a manner to reduce the structural constraint.

Circularly permuted, straight split L27V variants CP84 and CP103(N-(SS-169)-(1-SS₁)-FRB-C and C-(1-SS₁)-(SS-169)-FKBP) were cloned aspreviously described and expressed (25 μL) in rabbit reticulocyte lysate(RRL; Promega Corp.) following the manufacture's instructions. 1.25 μLof the expression reactions for each dimerization pair were mixed with10 μl, of 2× Binding Buffer (100 mM HEPES, 200 mM NaCl, 0.2% CHAPS, 2 mMEDTA, 20% glycerol, 20 mM DTT, pH 7.5) and 7.5 μL water, and 18 μLtransferred to wells of a 96-well plate. To the reactions, 2 μLrapamycin (final concentration 0 and 0.1-1000 nM) was added, and thereactions incubated at room temperature for 10 min. Followingincubation, 100 μL of PBI-3939 (50× stock diluted to 1× in assay buffer)and incubated for 3 min at room temperature. Luminescence was measuredon a GLOMAX® luminometer (FIG. 87B) and the response was determined(FIG. 87C). FIGS. 87B-C demonstrates that the OgLuc variants of thepresent invention can be used to detect protein-protein interactions viaa PCA-type dimerization assay.

Example 58 Intercellular Half-Life

The intracellular half-life of the OgLuc variants 9B8, 9B8+K33N, V2,L27V, and V2+L27M were determined. CHO cells (500,000) in 15-100 mmplates in F12 media with 10% FBS and 1× sodium pyruvate were transfectedwith 30 μL 100 ng/μL plasmid DNA containing 9B8, 9B8+K33N, V2, L27V(“V2+L27V”) or V2+L27M (all in pF4A vector background) usingTRANSIT®-LT1 (Mirus) according to the manufacture's instructions. Thecells were then incubated for 6 hrs.

After incubation, the media was removed and 1 mL Trypsin added todissociate the cells from the plate. 3 mL of F12 media was then added,and the cells counted. Cells were then plated at 10,000 cells/well into6 wells of a 96-well plate (6 wells/variant) and incubated overnight at37° C. Samples were distributed over 3 plates. Each plate had 6replicates for different time point measurements.

After overnight incubation, the media was removed from the cells for t=0samples, and 100 μL assay buffer (previously described; no substrate)was added. The sample was frozen on dry ice and stored at −20° C.Cycloheximide (100 mg/mL) was diluted 1:100 to a final concentration of1 mg/mL in OPTI-MEM®. DMSO (100%) was also diluted 1:100 (finalconcentration 1%) in OPTI-MEM®. The diluted cycloheximide (1 mg/mL) wasadded (11 μL) to 3 replicates of each transfected variant sample and 11μL of the diluted DMSO (1%) was added to the other 3 replicates. Thecells were then incubated at 37° C., 5% CO₂ and removed at varioustimepoints (i.e., 0, 0.5, 0.9, 2.5, 4.3, and 6.2 hrs) and processed asthe t=0 samples.

For analysis, the samples were thawed to room temperature, and 10 μLassayed in 50 μL assay reagent. Luminescence was measured on a GLOMAX®luminometer. At each time-point, luminescence was measured for untreatedand cycloheximide-treated samples. The RLU for the cells treated withcycloheximide was normalized by the RLU for the untreated cells.

The intracellular half-life of each variant was calculated by measuringthe ratio of the luminescence from the cycloheximide (CHX)-treated tothe untreated at each time-point. The ratio was then plotted ln (%treated to untreated) over time, and the half-life calculated (Table46). The OgLuc variants had intracellular half-lives of approximately6-9 hrs with a full strength CMV promoter, but the half lives werereduced with a CMV deletion variant (d2). The presence of a PESTdegradation signal combined with the full strength CMV promoter reduceshalf-life significantly.

TABLE 46 Sample CMV no deg. CMV d2 no deg. CMV Pest 9B8 6.32 3.87 1.43K33N 9.24 3.70 1.18 V2 9.63 4.28 1.61 V2 + L27V 6.66 4.78 1.63 V2 + L27M8.89 6.98 1.63

Another experiment was completed using the reverse transfection protocoldescribed in Example 52 with HEK293 cells (data not shown). The resultsfrom this experiment indicate that the intracellular half-life for theL27V variant with PEST is 10 min. The L27V variant with no degradationsignal used in this experiment did not show a decay over the course ofthis experiment. In this case the decay was normalized to untreatedcells at t=0.

Example 59 Exposure of OgLuc Variants to Urea

Since Firefly luciferase is known to be relatively unstable, it is muchmore sensitive to urea exposure. To determine whether this was also thecase with the OgLuc variants, the sensitivity of the OgLuc to urea wasdetermined. 5 μl of 45.3 μM L27V enzyme was mixed with 100 μL of a ureasolution (100 mM MOPS, pH 7.2, 100 mM NaCl, 1 mM CDTA, 5% glycerol andvarious concentrations of urea) and incubated for 30 min at roomtemperature. 5 μL of the urea+L27V enzyme solution was diluted10,000-fold into DMEM without phenol red+0.1% PRIONEX®, 50 μl was mixedwith 50 μL of assay reagent containing 100 μM PBI-3939 (previouslydescribed) and the luminescence was read at 10 min. (FIG. 88). FIG. 88indicates that L27V is either resistant to urea or refolds to afunctional enzyme very quickly upon removal of urea. This suggests thatL27V could be used as a reporter enzyme when chemical denaturingconditions are involved, e.g., multiplexing in conditions where adenaturant is used to stop an enzymatic reaction prior to the OgLucvariant-based reaction.

A 0.31 mg/mL stock of purified L27V variant was diluted 100,000-foldinto buffer (PBS+1 mM DTT+0.005% IGEPAL) and incubated with 3 M urea for30 min at 25° C. and then mixed 1:1 (50 μL+50 μL) with an assay reagentcontaining 100 μM PBI-3939 (previously described). The reactions wereread on a TECAN® INFINITE® F500 luminometer as described previously (for100 min; 1 min read intervals) (FIG. 89). The results indicate that 3Murea reduces the activity of L27V variant by approximately 50%, but,upon diluting out the urea by 2-fold (to a 1.5 M final concentration)the activity increases, presumably due to refolding.

Example 60 Imaging of OgLuc Fusion Proteins

This example demonstrates the use of OgLuc and OgLuc variants to monitorprotein translocation in living cells without the need for fluorescenceexcitation. OgLuc variants were fused to human glucocorticoid receptor(GR; SEQ ID NOs: 451 and 452), human protein kinase C alpha (PKCa; SEQID NOs: 449 and 450) or LC3 (SEQ ID NOs: 577 and 578). To analyzesubcellular protein translocation using bioluminescence imaging, HeLacells were plated at 2×10⁴ cells/cm² into glass-bottom culture dishes(MatTek) in DMEM medium (Invitrogen) containing 10% FBS. The cells werethen incubated for 24 hrs at 37° C., 5% CO2. Cells were then transfectedwith 1/20 volume transfection mixture (FUGENE® HD and DNA encodingL27V02-GR (SEQ ID NOs: 453 and 454) or L27V02-PKC alpha (SEQ ID NOs: 455and 456) cloned into the pF5A vector (Promega Corp.)). The plasmid DNAfor L27V02-GR was diluted 1:20 into pGEM-3ZF (Promega Corp.) to achieveappropriate expression levels of L27V02-GR. The plasmid DNA forL27V02-LC3 and L27V02-PKC alpha was used undiluted. Cells were thenincubated for 24 hrs at 37° C., 5% CO₂. Cells transfected with GR fusionproteins were starved of GR agonist for 20 hrs using MEM mediumsupplemented with 1% charcoal/dextran-treated FBS (Invitrogen).Twenty-four hrs post-transfection (for PKC alpha measurements) or 48 hrspost-transfection (for GR measurements), the cell media was replacedwith CO₂-independent media containing 100 μM PBI-3939 immediately beforeimaging. Unfiltered images were immediately captured on an Olympus LV200bioluminescence microscope using a 150× objective.

Cytosol-to-nucleus translocation of L27V02-GR fusion protein wasachieved via stimulation with 0.5 mM dexamethasone for 15 min.Cytosol-to-plasma membrane translocation of L27V02-PKC alpha fusionprotein was achieved via stimulation with 100 nM PMA for 20 min.L27V02-LC3 fusion protein transfected cells were left untreated ortreated with 50 mM Chloroquine in DMEM medium (Invitrogen) containing10% FBS.

L27V02-Glucocorticoid Receptor

In the absence of glucocorticoids, glucocorticoid receptor (GR) iscomplexed to Hsp90 proteins and resides in the cytosol. Upon interactionof GR with glucocorticoids, such as dexamethasone, GR proteinsdissociate from these protein complexes and translocate to the nucleusto regulate gene transcription. FIGS. 90A-B show the bioluminescenceimaging of dexamethasone-induced cytosol to nuclear receptor (NR)translocation of L27V02-glucocorticoid receptor (GR) fusion proteinsusing PBI-3939 substrate in HeLa cells.

L27V02-PKCa

Upon treatment with phorbol esters, PKC alpha proteins are recruited tothe plasma membrane and regulate cellular responses including membranedynamics and signal transduction. FIGS. 91A-B show the bioluminescenceimaging of phorbol ester-induced Protein Kinase C alpha (PKC alpha)cytosol to plasma membrane translocation of OgLuc L27V02-PKC alphafusions using PBI3939 substrate in U-20S cells.

L27V-LC3

Association of processed LC-3 proteins with autophagosomes represents ahallmark step in autophagy. Chloroquine treatment arrests autophagicflux at this stage, resulting in accumulation of LC-3 proteins onautophagosomes (producing a punctate subcellular distribution). FIGS.92A-B show the bioluminescence imaging of chloroquine-inducedautophagosomal protein translocation of OgLuc L27V-LC3 fusions proteins(SEQ ID NOs: 592 and 593) using PBI-3939 substrate in two representativeHeLa cell samples.

TABLE 47 Proviso List C1 + A4E Additional Substitutions C1 + A4E L92GC1 + A4E L92Q C1 + A4E L92S C1 + A4E L92A C1 + A4E L72Q C1 + A4E I90TC1 + A4E Q20R C1 + A4E C164S C1 + A4E M75K C1 + A4E V79I C1 + A4E F54IC1 + A4E K89E C1 + A4E I90V C1 + A4E F77W F68S I90V C1 + A4E F54I M75KC1 + A4E F54I M75K I90V C1 + A4E F54I F68S M75K C1 + A4E F54I I90V C1 +A4E F54I C1 + A4E F54T N135K I167V P104L D139E C1 + A4E V45E L34M G51VI99V I143L C1 + A4E S28P L34M G51V I99V I143L F54T C1 + A4E S28P L34MG51V I99V I143L F54T C1 + A4E S28P

1. A compound of formula (Ia) or (Ib):

wherein R² is selected from the group consisting of

or C₂₋₅ straight chain alkyl; R⁶ is selected from the group consistingof —H, —OH, —NH₂, —OC(O)R or —OCH₂OC(O)R; R⁸ is selected from the groupconsisting of

H or lower cycloalkyl; wherein R³ and R⁴ are both H or both C₁₋₂ alkyl;W is —NH₂, halo, —OH, —NHC(O)R, —CO₂R; X is —S—, —O— or —NR²²—; Y is —H,—OH, or —OR¹¹; Z is —CH— or —N—; each R¹¹ is independently —C(O)R″ or—CH₂OC(O)R″; R²² is H, CH₃ or CH₂CH₃; each R is independently C₁₋₇straight-chain alkyl or C₁₋₇ branched alkyl; R″ is C₁₋₇ straight-chainalkyl or C₁₋₇ branched alkyl; the dashed bonds indicate the presence ofan optional ring, which may be saturated or unsaturated; with theproviso that when R² is

or

R⁸ is not

with the proviso that when R² is

R⁸ is

or lower cycloalkyl; and with the proviso that when R⁶ is NH₂, R² is,

or C₂₋₅ alkyl; or R⁸ is not


2. A compound according to claim 1, wherein R² is

and X is O or S.
 3. A compound according to claim 1, wherein R² is C₂₋₅straight-chain alkyl.
 4. A compound according to claim 1, wherein R⁸ is

lower cycloalkyl or H.
 5. A compound according to claim 1, wherein R⁸ is


6. A compound according to claim 1, wherein R₁₁ is —CH₂OC(O)C(CH₃)₃. 7.A compound according to claim 1, wherein R″ is —C(CH₃)₃, —CH(CH₃)₂,—CH₂C(CH₃)₃, or —CH₂CH(CH₃)₂.
 8. A compound selected from


9. A compound of formula


10. A kit comprising a compound according to claim
 1. 11. The kit ofclaim 10 further comprising a luciferase.
 12. The kit of claim 11,wherein the luciferase is an Oplophorus or Renilla luciferase.
 13. Thekit of claim 10, further comprising a buffer reagent.
 14. A method fordetecting luminescence in a sample comprising contacting a sample with acompound according to claim 1; contacting the sample with acoelenterazine-utilizing luciferase, if it is not present in the sample;and detecting luminescence.
 15. The method of claim 14, wherein thesample contains live cells.
 16. The method of claim 14, wherein thesample contains a coelenterazine-utilizing luciferase.
 17. A method fordetecting luminescence in a transgenic animal comprising administering acompound according to claim 1 to a transgenic animal; and detectingluminescence; wherein the transgenic animal expresses acoelenterazine-utilizing luciferase.